Anti-VEGF antibodies

ABSTRACT

Anti-VEGF antibodies and variants thereof, including those having high affinity for binding to VEGF, are disclosed. Also provided are methods of using phage display technology with naïve libraries to generate and select the anti-VEGF antibodies with desired binding and other biological activities. Further contemplated are uses of the antibodies in research, diagnostic and therapeutic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/414,742, filed on Apr. 27, 2006, now U.S. Pat. No. 7,758,859, whichis a continuation of International Application No. PCT/US2004/029332,filed on Sep. 8, 2004, which claims benefit from U.S. ProvisionalApplication Nos. 60/491,877, filed Aug. 1, 2003; 60/516,495, filed Nov.1, 2003; 60/570,912, filed May 12, 2004; 60/571,239, filed May 13, 2004;60/576,315, filed Jun. 1, 2004; and 60/580,757, filed Jun. 18, 2004.U.S. patent application Ser. No. 11/414,742 is also acontinuation-in-part of U.S. application Ser. No. 11/342,249, filed Jan.27, 2006, now U.S. Pat. No. 7,811,785, and a continuation-in-part ofInternational Application No. PCT/US2004/024662, filed Jul. 30, 2004,which claims benefit from U.S. Provisional Application Nos. 60/491,877,filed Aug. 1, 2003; 60/516,495, filed Nov. 1, 2003; 60/570,912, filedMay 12, 2004; 60/571,239, filed May 13, 2004; 60/576,315, filed Jun. 1,2004; and 60/580,757, filed Jun. 18, 2004. All of these applications arehereby incorporated by reference in their entirety.

REFERENCE TO A COMPUTER PROGRAM LISTING APPENDIX

A Sequence Listing is provided in this patent document as a txt fileentitled, “50474.016007 Sequence Listing_ST25.txt,” created May 5, 2009(size: 709 KB).

FIELD OF THE INVENTION

This invention relates generally to anti-VEGF selected polypeptidesequences and antibodies with beneficial properties for research,therapeutic and diagnostic purposes.

BACKGROUND OF THE INVENTION Angiogenesis and VEGF

Angiogenesis is an important cellular event in which vascularendothelial cells proliferate, prune and reorganize to form new vesselsfrom preexisting vascular networks. There are compelling evidences thatthe development of a vascular supply is essential for normal andpathological proliferative processes (Folkman and Klagsbrun (1987)Science 235:442-447). Delivery of oxygen and nutrients, as well as theremoval of catabolic products, represent rate-limiting steps in themajority of growth processes occurring in multicellular organisms. Thus,it has been generally assumed that the vascular compartment isnecessary, albeit but not sufficient, not only for organ development anddifferentiation during embryogenesis, but also for wound healing andreproductive functions in the adult.

Angiogenesis is also implicated in the pathogenesis of a variety ofdisorders, including but not limited to, proliferative retinopathies,age-related macular degeneration, tumors, rheumatoid arthritis (RA), andpsoriasis. Angiogenesis is a cascade of processesconsisting of 1)degradation of the extracellular matrix of a local venue after therelease of protease, 2) proliferation of capillary endothelial cells,and 3) migration of capillary tubules toward the angiogenic stimulus.Ferrara et al. (1992) Endocrine Rev. 13:18-32.

In view of the remarkable physiological and pathological importance ofangiogenesis, much work has been dedicated to the elucidation of thefactors capable of regulating this process. It is suggested that theangiogenesis process is regulated by a balance between pro- andanti-angiogenic molecules, and is derailed in various diseases,especially cancer. Carmeliet and Jain (2000) Nature 407:249-257.

Vascular endothelial cell growth factor (VEGF), a potent mitogen forvascular endothelial cells, has been reported as a pivotal regulator ofboth normal and abnormal angiogenesis. Ferrara and Davis-Smyth (1997)Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol. Med. 77:527-543. Comparedto other growth factors that contribute to the processes of vascularformation, VEGF is unique in its high specificity for endothelial cellswithin the vascular system. Recent evidence indicates that VEGF isessential for embryonic vasculogenesis and angiogenesis. Carmeliet etal. (1996) Nature 380:435-439; Ferrara et al. (1996) Nature 380:439-442.Furthermore, VEGF is required for the cyclical blood vesselproliferation in the female reproductive tract and for bone growth andcartilage formation. Ferrara et al. (1998) Nature Med. 4:336-340; Gerberet al. (1999) Nature Med. 5:623-628.

In addition to being an angiogenic factor in angiogenesis andvasculogenesis, VEGF, as a pleiotropic growth factor, exhibits multiplebiological effects in other physiological processes, such as endothelialcell survival, vessel permeability and vasodilation, monocyte chemotaxisand calcium influx. Ferrara and Davis-Smyth (1997), supra. Moreover,recent studies have reported mitogenic effects of VEGF on a fewnon-endothelial cell types, such as retinal pigment epithelial cells,pancreatic duct cells and Schwann cells. Guerrin et al. (1995) J. CellPhysiol. 164:385-394; Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol.126:125-132; Sondell et al. (1999) J. Neurosci. 19:5731-5740.

Substantial evidence also implicates VEGF's critical role in thedevelopment of conditions or diseases that involve pathologicalangiogenesis. The VEGF mRNA is overexpressed by the majority of humantumors examined (Berkman et al. J Clin Invest 91:153-159 (1993); Brownet al. Human Pathol. 26:86-91 (1995); Brown et al. Cancer Res.53:4727-4735 (1993); Mattern et al. Brit. J. Cancer. 73:931-934 (1996);and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)). Also, theconcentration of VEGF in eye fluids are highly correlated to thepresence of active proliferation of blood vessels in patients withdiabetic and other ischemia-related retinopathies (Aiello et al. N.Engl. J. Med. 331:1480-1487 (1994)). Furthermore, recent studies havedemonstrated the localization of VEGF in choroidal neovascular membranesin patients affected by AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci.37:855-868 (1996)).

The recognition of VEGF as a primary regulator of angiogenesis inpathological conditions has led to numerous attempts to block VEGFactivities Inhibitory anti-VEGF receptor antibodies, soluble receptorconstructs, antisense strategies, RNA aptamers against VEGF and lowmolecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have allbeen proposed for use in interfering with VEGF signaling (Siemeister etal. Cancer Metastasis Rev. 17:241-248 (1998). Indeed, anti-VEGFneutralizing antibodies have been shown to suppress the growth of avariety of human tumor cell lines in nude mice (Kim et al. Nature362:841-844 (1993); Warren et al. J. Clin. Invest. 95:1789-1797 (1995);Borgström et al. Cancer Res. 56:4032-4039 (1996); and Melnyk et al.Cancer Res. 56:921-924 (1996)) and also inhibit intraocular angiogenesisin models of ischemic retinal disorders (Adamis et al. Arch. Ophthalmol.114:66-71 (1996)). Therefore, anti-VEGF monoclonal antibodies or otherinhibitors of VEGF action are promising candidates for the treatment ofsolid tumors and various intraocular neovascular disorders. Although theVEGF molecule is upregulated in tumor cells and its receptors areupregulated in tumor infiltrated vascular endothelial cells, theexpression of VEGF and its receptors remain low in normal cells that arenot associated with angiogenesis.

Therapeutic Antibodies

Monoclonal antibodies can be manufactured using recombinant DNAtechnology. Widespread use has been made of monoclonal antibodies,particularly those derived from rodents; however, nonhuman antibodiesare frequently antigenic in humans. The art has attempted to overcomethis problem by constructing “chimeric” antibodies in which a nonhumanantigen-binding domain is coupled to a human constant domain (Cabilly etal., U.S. Pat. No. 4,816,567). The isotype of the human constant domainmay be selected to tailor the chimeric antibody for participation inantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity. In a further effort to resolve the antigen bindingfunctions of antibodies and to minimize the use of heterologoussequences in human antibodies, humanized antibodies have been generatedfor various antigens in which substantially less than an intact humanvariable domain has been substituted at regions by the correspondingsequence from a non-human species. For example, rodent (CDR) residueshave been substituted for the corresponding segments of a humanantibody. In practice, humanized antibodies are typically humanantibodies in which some complementarity determining region (CDR)residues and possibly some framework region (FR) residues aresubstituted by residues from analogous sites in rodent antibodies. Joneset al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327(1988); Verhoeyen et al., Science 239:1534-1536 (1988).

Several humanized anti-humanVEGF antibodies have been successfullygenerated, and have shown significant hVEGF-inhibitory activities bothin vitro and in vivo. Presta et al. (1997) Cancer Research 57:4593-4599;Chen et al. (1999) J. Mol. Biol. 293:865-881. One specific humanizedanti-VEGF antibody, the Avastin™ antibody, is currently used in severalclinical trials for treating various solid tumors; and anotherhigh-affinity variant of the humanized anti-VEGF antibody is currentlyclinically tested for treating choroidal neovascularization related agemacular degeneration (AMD).

Prior to administering a therapeutic antibody to human, preclinicalstudies in nonhuman mammals are generally desired to evaluate theefficacy and/or toxicity of the antibody. Ideally, the antibodiessubject to these studies are capable of recognizing and reacting withhigh potency to a target antigen endogenous to the host animal such asmouse or nonhuman primate.

Phage Display

Phage display technology has provided a powerful tool for generating andselecting novel proteins that bind to a ligand, such as an antigen.Using the technique of phage display, large libraries of proteinvariants can be generated and rapidly sorted for those sequences thatbind to a target antigen with high affinity. Nucleic acids encodingvariant polypeptides are fused to a nucleic acid sequence encoding aviral coat protein, such as the gene III protein or the gene VIIIprotein. Monovalent phage display systems where the nucleic acidsequence encoding the protein or polypeptide is fused to a nucleic acidsequence encoding a portion of the gene III protein have been developed.(Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: ACompanion to Methods in Enzymology, 3:205 (1991)). In a monovalent phagedisplay system, the gene fusion is expressed at low levels and wild typegene III proteins are also expressed so that infectivity of theparticles is retained. Methods of generating peptide libraries andscreening those libraries have been disclosed in many patents (e.g.,U.S. Pat. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No.5,580,717, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,498,530).

The demonstration of expression of peptides on the surface offilamentous phage and the expression of functional antibody fragments inthe periplasm of E. coli was important in the development of antibodyphage display libraries. (Smith et al., Science (1985), 228:1315; Skerraand Pluckthun, Science (1988), 240:1038). Libraries of antibodies orantigen binding polypeptides have been prepared in a number of waysincluding by altering a single gene by inserting random DNA sequences orby cloning a family of related genes. Methods for displaying antibodiesor antigen binding fragments using phage display have been described inU.S. Pat. Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197,5,580,717, and 5,658,727. The library is then screened for expression ofantibodies or antigen binding proteins with desired characteristics.

Phage display technology has several advantages over conventionalhybridoma and recombinant methods for preparing antibodies with thedesired characteristics. This technology allows the development of largelibraries of antibodies with diverse sequences in less time and withoutthe use of animals. Preparation of hybridomas or preparation ofhumanized antibodies can easily require several months of preparation.In addition, since no immunization is required, phage antibody librariescan be generated for antigens which are toxic or have low antigenicity(Hogenboom, Immunotechniques (1988), 4:1-20). Phage antibody librariescan also be used to generate and identify novel therapeutic antibodies.

Phage display libraries have been used to generate human antibodies fromimmunized humans, non-immunized humans, germ line sequences, or naïve Bcell Ig repertories (Barbas & Burton, Trends Biotech (1996), 14:230;Griffiths et al., EMBO J. (1994), 13:3245; Vaughan et al., Nat. Biotech.(1996), 14:309; Winter EP 0368 684 B1). Naïve, or nonimmune, antigenbinding libraries have been generated using a variety of lymphoidaltissues. Some of these libraries are commercially available, such asthose developed by Cambridge Antibody Technology and Morphosys (Vaughanet al. (1996) Nature Biotech 14:309; Knappik et al. (1999) J. Mol. Biol.296:57). However, many of these libraries have limited diversity.

The ability to identify and isolate high affinity antibodies from aphage display library is important in isolating novel antibodies fortherapeutic use. Isolation of high affinity antibodies from a library isdependent on the size of the library, the efficiency of production inbacterial cells and the diversity of the library. See, for e.g., Knappiket al., J. Mol. Biol. (1999), 296:57. The size of the library isdecreased by inefficiency of production due to improper folding of theantibody or antigen binding protein and the presence of stop codons.Expression in bacterial cells can be inhibited if the antibody orantigen binding domain is not properly folded. Expression can beimproved by mutating residues in turns at the surface of thevariable/constant interface, or at selected CDR residues. (Deng et al.,J. Biol. Chem. (1994), 269:9533, Ulrich et al., PNAS (1995),92:11907-11911; Forsberg et al., J. Biol. Chem. (1997), 272:12430). Thesequence of the framework region is a factor in providing for properfolding when antibody phage libraries are produced in bacterial cells.

Generating a diverse library of antibodies or antigen binding proteinsis also important for isolation of high affinity antibodies. Librarieswith diversification in limited CDRs have been generated using a varietyof approaches. See, for e.g., Tomlinson, Nature Biotech. (2000),18:989-994. CDR3 regions are of interest in part because they often arefound to participate in antigen binding. CDR3 regions on the heavy chainvary greatly in size, sequence and structural conformation.

Others have also generated diversity by randomizing CDR regions of thevariable heavy and light chains using all 20 amino acids at eachposition. It was thought that using all 20 amino acids would result in alarge diversity of sequences of variant antibodies and increase thechance of identifying novel antibodies. (Barbas, PNAS 91:3809 (1994);Yelton, D E, J. Immunology, 155:1994 (1995); Jackson, J. R., J.Immunology, 154:3310 (1995) and Hawkins, R E, J. Mol. Biology, 226:889(1992)).

There have also been attempts to create diversity by restricting thegroup of amino acid substitutions in some CDRs to reflect the amino aciddistribution in naturally occurring antibodies. See, Garrard & Henner,Gene (1993), 128:103; Knappik et al., J. Mol. Biol. (1999), 296:57.However, these attempts have had varying success and have not beenapplied in a systematic and quantitative manner. Creating diversity inthe CDR regions while minimizing the number of amino acid changes hasbeen a challenge.

SUMMARY OF THE INVENTION

The present invention provides novel antibodies and polypeptidesequences. The present invention also provides antibodies capable ofbinding to rodent VEGF and human VEGF with Kd values within 10 fold ofeach value and are capable of inhibiting the binding of VEGF to a VEGFreceptor. According to one embodiment, the antibody is capable ofbinding to either one or both of a Gly88Ala (G88A) or a Gly88Ser (G88S)human VEGF mutant with a Kd value that is within 10 fold of the Kd valueof unmutated human VEGF.

The present invention provides antibodies that are capable of binding toa human VEGF and a mouse VEGF with Kd values that are 10 nM or less at25° C. and are capable of inhibiting the binding of VEGF to a VEGFreceptor. According to another embodiment the Kd values are 2 nM orless. According to another embodiment, the Kd values are 0.1 nM or less.According to another embodiment, an antibody of this invention bindsVEGF with a Kd of no more than about 1 nM, or no more than about 500 pM.

The present invention also provides antibodies that are capable ofbinding to a human VEGF and to either one or both G88A and G88S mutantsof human VEGF with Kd values that are 10 nM or less and are capable ofinhibiting the binding of VEGF to a VEGF receptor. In anotherembodiment, the antibodies bind to a human VEGF and to either one orboth human VEGF G88A and G88S mutants with Kd values that are 10 nM orless.

According to another embodiment, an antibody of this invention has anon-rate (k_(on)) for binding to human and/or mouse VEGF that is 1.0 ormore (10 M⁻¹ S⁻¹). According to another embodiment, the on-rate is 5.0or more (10 M⁻¹ S⁻¹). According to yet another embodiment, the on-rateis 10.0 or more (10 M⁻¹ S⁻¹).

According to another embodiment, the antibodies of this inventioncontact less than 80% of the total surface area (angstrom²) of G88 ofhuman VEGF. According to another embodiment, the antibodies of thisinvention contact less than 70% of the total surface area (angstrom²) ofG88 of human VEGF. According to another embodiment, the antibodies ofthis invention contact less than 60% of the total surface area(angstrom²) of G88 of human VEGF. According to another embodiment, theantibodies of this invention contact less than 1% of the total surfacearea (angstrom²) of G88 of human VEGF. Such antibodies are generallycapable of also binding mouse VEGF.

The VEGF receptor to be inhibited from binding to VEGF can be the VEGFreceptor 1 (Flt-1), VEGF receptor 2 (Flt-1) or both receptors.

According to one embodiment, an antibody of this invention contacts the20 s helix of VEGF. According to another embodiment, an antibody of thisinvention contacts the 80 s loop of VEGF. According to yet anotherembodiment, an antibody of this invention contacts the 20 s helix andthe 80 s loop of human VEGF.

According to one embodiment, an antibody of this invention has relativeaffinity for or is capable of contacting any one of residues F17, M18,Q22, Y25, D63, L66, C104 and P106 of human VEGF. According to anotherembodiment, an antibody of this invention has relative affinity for oris capable of contacting F17, M18, Q22, Y25, D63, L66, C104 and P106 ofhuman VEGF. According to another embodiment, an antibody of thisinvention has relative affinity for or is capable of contacting residuesF17 and Y21 of human VEGF. According to another embodiment, an antibodyof this invention has further relative affinity for or is capable ofcontacting Y25 of VEGF. According to another embodiment, an antibody ofthis invention has relative affinity for or is capable of contacting M18and Q89 human VEGF. According to another embodiment, an antibody of thisinvention has relative affinity for or is capable of contacting M18, Y21and Y25 of human VEGF. According to a preferred embodiment, an antibodyof this invention has a combination of three or more of any one of theabove-mentioned embodiments.

In a further embodiment, an antibody of this invention has a functionalepitope described herein. According to one embodiment, the functionalepitope of an antibody according to this invention includes residue F17of human VEGF. According to another embodiment, the functional epitopeof an antibody according to this invention includes residues F17 and 183of human VEGF. According to a further embodiment, the functional epitopeincludes residues F17, I83 and Q89 of human VEGF. According to oneembodiment, the functional epitope of an antibody according to thisinvention includes residues F17 and M18 of human VEGF. In a furtherembodiment, the functional epitope includes residues F17, M18 and 189 ofhuman VEGF. According to one embodiment, the functional epitope of anantibody according to this invention includes residues F17, Y21 and Y25of human VEGF. According to a further embodiment, the functional epitopeincludes residues F17, Y21, Q22, Y25, D63 and 183 of human VEGF.According to an alternative embodiment, the functional epitope includesresidues F17, Y21, Y25 and Q89 of human VEGF. According to a furtherembodiment, the functional epitope includes residues F17, M18, D19, Y21,Y25, Q89, I91, K101, E103, and C104 of human VEGF. According to yetanother embodiment, the functional epitope includes residues 17, Y21,Q22, Y25, D63, I83 and Q89 of human VEGF. A functional epitope of anantibody according to this invention can include a combination of any ofthe residues selected from the group consisting of F17, M18, Y21, Q22,Y25, K48, D63, L66, M81, I83, H86, Q89, I91, C104 and P106 of humanVEGF. According to another embodiment, the functional epitope includesresidues F17, M18, Y21, Q22, Y25, K48, D63, L66, M81, I83, H86, Q89,I91, C104 and P106 of human VEGF.

According to one embodiment, an antibody of this invention comprises aCDR-H3 comprising the contiguous amino acid sequence X₁X₂FX₄X₅X₆X₇ (SEQID NO: 915), wherein

X₁ is Y or F;

X₂ is V or A;

X₄ is F or Y;

X₅ is L or A;

X₆ is P or A; and

X₇ is Y or F.

According to another embodiment, the antibody further comprises a CDR-H2having a contiguous amino acid sequence GX₂TPX₅G (SEQ ID NO: 1), wherein

X₂ is a I or V or other hydrophobic amino acid; and

X₅ is any amino acid residue.

According to yet another embodiment, the antibody still furthercomprises a CDR-H1 having a contiguous amino acid sequence X₁X₂X₃₁H (SEQID NO: 2), wherein

X₁ is any amino acid residue;

X₂ is Y or F; and

X₃ is W or L.

According to one preferred embodiment, the antibody further comprisesthe CDR-L1, CDR-L2 and CDR-L3 of any one of the antibodies of FIG. 7.According to another embodiment, the CDR-L1 is located at approximatelyresidues 28-33, the CDR-L2 is located at approximately residues 50-55;and the CDR-L3 is located at approximately residues 91-96. According toanother embodiment, the CDR-H1 is located at approximately residues30-33, the CDR-H2 is located at approximately residues 50-58; and theCDR-L3 is located at approximately residues 94-100a. According to onepreferred embodiment, the antibody comprises the framework regions ofthe G6 antibody, the G6-23 antibody or the G6-31 antibody. According toanother embodiment, the antibody comprises the light chain CDRs or thelight chain variable region of the G6 antibody, the G6-23 antibody orthe G6-31 antibody.

According to one embodiment, an antibody of this invention is anantibody comprising:

(a) a CDR-L1 comprising the contiguous amino acid sequence X₁X₂X₃X₄X₅L(SEQ ID NO: 916), wherein:

X₁ and X₂ are any amino acid;

Either X₃ or X₄ or both X₃ and X₄ are R;

X₅ is S or A; and

(b) a CDR-L2 comprising a contiguous amino acid sequence X₁X₂X₃ (SEQ IDNO: 917), wherein

X₁ is S or A or G; and

X₂ and X₃ are any amino acid residue; and

(c) a CDR-L3 comprising a contiguous amino acid sequence SX₁X₂X₃PL (SEQID NO: 918), wherein

X₁ and X₂ are any amino acid residue; and

X₃ is S or A.

According to one preferred embodiment, the antibody comprises theframework regions of the B20-4.1 antibody or the B20-4 antibody.According to another embodiment, the antibody comprises the CDRs or thevariable region of the B20 heavy chain variable region. According toanother embodiment, the X₁X₂X₃ (SEQ ID NO: 917) of the CDR-L2 is encodedby X₁ASX₄LX₆ (SEQ ID NO: 919), wherein X₄ and X₆ are any amino acid.According to another embodiment, the CDR-L1 is located at approximatelyresidues 28-33, the CDR-L2 is located at approximately residues 50-55,and the CDR-L3 is located at approximately residues 91-96. According toanother embodiment, the CDR-H1 is located at approximately residues30-33, the CDR-H2 is located at approximately residues 50-58, and theCDR-L3 is located at approximately residues 94-100a.

Also contemplated is an antibody comprising a CDR-H3 sequence of any oneof the antibodies of FIGS. 7, 24-29 and 34-43 and optionally furthercomprising a CDR-H2 and/or a CDR-H1 of the same antibody from FIGS. 7,24-29 and 34-43. Also, contemplated is an antibody selected from thegroup consisting of a G6 series antibody, a B20 series antibody, YADSseries antibody and a YS series antibody. Further, another antibody ofthis invention is an antibody comprising a variable region of any one ofthe antibodies of FIGS. 7, 24-29 and 34-43.

According to one preferred embodiment, the antibodies of this inventionare synthesized by recombinant methods rather than produced directlyfrom a hybridoma or derived from an antibody sequence from a hybridoma.In one preferred embodiment, the antibody binds to hVEGF with a Kd valueof no more than about 2 nM, no more than about 1 nM, or no more thanabout 500 pM. According to one embodiment, the antibody is a monoclonalantibody. According to another embodiment, the antibody is amulti-specific antibody (e.g., a bispecific antibody).

In a further embodiment of the invention, the high affinity anti-hVEGFantibody is also capable of binding to a VEGF from a non-human mammalspecies with Kd values comparable to, or at least within ten-fold of,the Kd value for its hVEGF binding. Such antibodies with cross-specieshigh binding affinities are particularly useful for preclinical researchas well as diagnostic and therapeutic applications. Some of theantibodies intended for therapeutic use were generated using a targethuman antigen such as the immunogen. The resultant antibody may be“species-dependent”, i.e. while binding specifically to human antigen,it may be much less effective at binding a homologue of that antigenfrom a nonhuman mammal. This is found herein to be problematic,particularly where the nonhuman mammal is one in which preclinicalstudies of the antibody are to be carried out. An example is theanti-VEGF antibody, the Avastin™ antibody, used for treating cancers.While the Avastin™ antibody had a strong binding affinity for human VEGF(i.e. K_(d) 1.8 nM), the affinity for murine VEGF was unsuitable forconducting experiments in mouse models.

In one aspect, the antibodies are synthetic antibodies comprising atleast one variant CDR in its variable domains, wherein the variant CDRcomprises variant amino acid in at least one solvent accessible andhighly diverse amino acid position, wherein the variant amino acid isencoded by a nonrandom codon set, and wherein at least 70% of the aminoacids encoded by the nonrandom set are target amino acids for thatposition in known antibody variable domains. The antibody may have aheavy chain variable domain which comprises at least 1, 2 or 3 variantCDRs selected from the group consisting of CDR H1, H2 and H3. Preferablythe heavy chain variable domain comprises a variant CDR H3. The antibodymay also have a light chain variable domain which comprises at least 1,2 or 3 variant CDRs selected from the group consisting of CDR L1, L2 andL3.

According to another embodiment, an antibody of this invention bindshuman VEGF and a rodent VEGF with a desired affinity but does not bindto any one or all of the VEGF-related homologues selected from the groupconsisting of human VEGF-B, human VEGF-D, and human P1GF-2.

According to one preferred embodiment, an antibody of this invention hasa combination of three or more of any one of the above-mentionedembodiments. According to another embodiment, a Fab antibody of thisinvention is conjugated to an agent that will increase the half-life ofthe Fab antibody. According to one preferred embodiment, the agent is apolypeptide comprising the sequence DICLPRWGCLW (SEQ ID NO:935). In apreferred embodiment, the antibodies of this invention do not bind toP1GF, VEGF-D or VEGF-B.

The invention also provide a method of selecting a hVEGF antibody from alibrary of synthetic antibodies comprising: a) generating the library ofsynthetic antibodies having a designed diversity in at least one of theCDRs; b) contacting the library with hVEGF to form binders; c)separating the binders from the nonbinders, and eluting the binders fromthe target hVEGF and incubating the binders in a solution withdecreasing amounts of the target hVEGF in a concentration from about 0.1nM to 1000 nM; and c) selecting the binders that can bind to the lowestconcentration of the target VEGF and that have an affinity of about 500pM to 2 nM.

Also contemplated are variants of the synthetic antibodies with improvedbinding affinities to hVEGF or to VEGF of non-human species, or both.Various forms of the antibody and variants thereof are contemplatedherein. For example, the antibody mutant may be a full length antibody(e.g. having a human immunoglobulin constant region) or an antibodyfragment (e.g. a Fab or F(ab′)₂). Furthermore, the antibody mutant maybe labeled with a detectable label, immobilized on a solid phase and/orconjugated with a heterologous compound (such as a cytotoxic agent).

Diagnostic and therapeutic uses for the antibody are contemplated. Inone diagnostic application, the invention provides a method fordetermining the presence of a protein of interest comprising exposing asample suspected of containing the protein to the antibody mutant anddetermining binding of the antibody mutant to the sample. For this use,the invention provides a kit comprising the antibody mutant andinstructions for using the antibody mutant to detect the protein.

The invention further provides: an isolated nucleic acid encoding theantibody; a vector comprising the nucleic acid, optionally, operablylinked to control sequences recognized by a host cell transformed withthe vector; a host cell transformed with the vector; a process forproducing the antibody comprising culturing this host cell so that thenucleic acid is expressed and, optionally, recovering the antibodymutant from the host cell culture (e.g. from the host cell culturemedium).

The invention also provides a composition comprising the antibody and apharmaceutically acceptable carrier or diluent. This composition fortherapeutic use is sterile and may be lyophilized. Also contemplated isthe use of an antibody or polypeptide of this invention in themanufacture of a medicament for treating an indication described herein.The composition can further comprise a second therapeutic agent such asa chemotherapeutic agent, a cytotoxic agent or an anti-angiogenic agent.

The invention further provides a method for treating a mammal,comprising administering an effective amount of the antibody to themammal. The mammal to be treated in the method may be a nonhuman mammal,e.g. a primate suitable for gathering preclinical data or a rodent(e.g., mouse or rat or rabbit). The nonhuman mammal may be healthy (e.g.in toxicology studies) or may be suffering from a disorder to be treatedwith the antibody of interest. In one embodiment, the mammal issuffering from or is at risk of developing abnormal angiogenesis (e.g.,pathological angiogenesis). In one specific embodiment, the disorder isa cancer selected from the group consisting of colorectal cancer, renalcell carcinoma, ovarian cancer, lung cancer, non-small-cell lung cancer(NSCLC), bronchoalveolar carcinoma and pancreatic cancer. In anotherembodiment, the disorder is a disease caused by ocularneovascularisation, e.g., diabetic blindness, retinopathies, primarilydiabetic retinopathy, age-induced macular degeneration and rubeosis. Inanother embodiment, the mammal to be treated is suffering from or is atrisk of developing an edema (e.g., an edema associated with braintumors, an edema associated with stroke, or a cerebral edema). Inanother embodiment, the mammal is suffering from or at risk ofdeveloping a disorder or illness selected from the group consisting ofrheumatoid arthritis, inflammatory bowel disease, refractory ascites,psoriasis, sarcoidosis, arterial arteriosclerosis, sepsis, burns andpancreatitis. According to another embodiment, the mammal is sufferingfrom or is at risk of developing a genitourinary illness selected fromthe group consisting of polycystic ovarian disease (POD), endometriosisand uterine fibroids. The amount of antibody administered will be atherapeutically effective amount to treat the disorder. In doseescalation studies, a variety of doses of the antibody may beadministered to the mammal. In another embodiment, a therapeuticallyeffective amount of the antibody is administered to a human patient totreat a disorder in that patient. In one preferred embodiment,antibodies of this invention useful for treating inflammatory or immunediseases described herein (e.g., rheumatoid arthritis) are Fab or scFvantibodies, especially Fab or scFv antibodies derived from the G6 seriesof antibodies or the B20 series of antibodies. Antibodies that do notcause aggregation of VEGF and do not aggregate themselves such as theB20 series of IgG antibodies are particularly useful in treatinginflammatory and immune diseases. Accordingly, such antibodies can beused in the manufacture of a medicament for treating an inflammatory orimmune disease. A mammal that is suffering from or is at risk fordeveloping a disorder or illness described herein can be treated byadministering, a second therapeutic agent, simultaneously, sequentiallyor in combination with, an antibody of this invention. It should beunderstood that other therapeutic agents, in addition to the secondtherapeutic agent, can be administered to the mammal or used in themanufacture of a medicament for the desired indications.

These antibodies and polypeptides can be used to understand the role ofhost stromal cell collaboration in the growth of implanted non-hosttumors, such as in mouse models wherein human tumors have beenimplanted. These antibodies and polypeptides can be used in methods ofidentifying human tumors that can escape anti-VEGF treatment byobserving or monitoring the growth of the tumor implanted into a rodentor rabbit after treatment with an anti-VEGF antibody of this invention.The antibodies and polypeptides of this invention can also be used tostudy and evaluate combination therapies with anti-VEGF antibodies ofthis invention and other therapeutic agents. The antibodies andpolypeptides of this invention can be used to study the role of VEGF inother diseases by administering the antibodies or polypeptides to ananimal suffering from a similar disease and determining whether one ormore symptoms of the disease are alleviated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of various types of antibodyfragments useful for phage display.

FIG. 2 compares residue changes within heavy chain CDRs of the templateantibody h4D5 and the four selected novel synthetic antibodies (SEQ IDNO: 921-928). Also compared are VEGF bindings of the four antibodies.

FIGS. 3A and 3B depict the abilities of VEGF receptors (Flt1-d2 and KDR)to block VEGF bindings of the novel anti-VEGF antibodies. Y0959A, ananti-VEGF variant, was used as a control.

FIG. 4 shows that the G6 antibody specifically binds to both hVEGF andmVEGF, but not to VEGF-related antigens.

FIGS. 5A and 5B show that G6 can effectively block both hVEGF and mVEGFbinding to the KDR receptor. Fab-12 and Y0317 were used as controls.

FIGS. 6A-6C show effects of G6 on VEGF-induced HUVEC proliferation.

FIGS. 7A and 7B illustrate the steps of generating high affinityanti-VEGF antibodies and the list of residues and binding affinitiesrelated to various affinity-improved G6 variants (SEQ ID NO: 44-84).

FIGS. 8A-8C compare VEGF bindings of G6, G6-23 and Fab-12. Associationrates (on-rate) to both hVEGF and mVEGF were measured over time; thecalculated Kon, Koff and Kd are listed.

FIGS. 9A and 9B compare VEGF binding on-rates of G6 and further improvedvariants of G6-23.

FIGS. 10A and 10B depict the effects of VEGF antagonists (G6-23 andFlt1-3Fc) on neonate mice body weights and survival rates.

FIGS. 11A and 11B depict the effects of G6-23 on tumor growth in nudemice with xenografted human tumor cells (KM12 cells and SW480 cells), asmeasured by tumor volumes over number of treatment days.

FIGS. 12A and 12B show VEGF expressions (both hVEGF and mVEGF) in KM12xenograft mice in the presence or absence of G6-23.

FIG. 13 depicts effects of VEGF antagonists (G6-23 and Flt1-3Fc) ontumor growth in nude mice with xenografted mouse tumor (LL2), asmeasured by tumor volumes over number of treatment days.

FIGS. 14A and 14B describe codons designed for shotgun scanning codonsof G6 and G6-23. For each scan, degenerate shotgun codons for (A) heavyand (B) light chain were designed to encode the wild-type amino acid andalanine (ml) in alanine-scan or similar amino acid (m4) in homolog-scanfor both G6 and G6-23 Fab. The shotgun codons are represented by the IUBcode (K=G/T, M=A/C, R=A/G, S=G/C, V=A/C/G, W=A/T, Y=C/T). Moresubstitutions (m2, m3, and m5) occurred in some residues due to thenature of the genetic code. In the case of wild-type alanine, theshotgun codon was designed to encode alanine and glycine. Each residueon CDRs is denoted by the single-letter code for amino acid, followed bya number denoting its position according to the scheme of Kabat et al.,1987. Positions where the sequences of G6 and G6-23 differ were shown intwo letters, e.g. Q89K denotes position 89 which is Gln or Lys in G6 orG6-23, respectively. Asterisks (*) indicate both alanine- andhomolog-scan codons encode a common substitution.

FIG. 15 provides information regarding the construction of G6 and G6-23shotgun scanning libraries. The shotgun scanning libraries wereperformed to mutate codons for the indicated residues on the heavy (hc)or light (lc) chain of G6 and G6-23 with either alanine- or homolog-scanshotgun codons (FIGS. 14A and B). In total, eight libraries wereconstructed using the mutagenic oligonucleotides as shown in Example 1.The theoretical diversity of each library and total numbers of aminoacid combinations encoded by the mutagenic oligonucleotides, wereexceeded at least 100-fold by the actual diversity of the constructedlibrary.

FIGS. 16A and 16B describe the results of the G6 and G6-23 heavy chainshotgun scan. The effect of each mutation (FIGS. 14A and B) on the heavychain CDR residues of (A) G6 and (B) G6-23 was evaluated by calculatingwt/mut ratios from sequences of functional clones, either from thealanine-scan libraries (m1, m2, and m3) or the homolog-scan libraries(m4 and m5), isolated after binding selection to either hVEGF (targetselection) or an anti-gD tag antibody (display selection). To provide aquantitative estimate of the effect of each mutation on the bindingaffinity of G6 and G6-23 for hVEGF, the function ratio (Fwt/mut) at eachmutation site was derived from dividing the wt/mut ratio from targetselection by that from display selection. Deleterious effects areindicated by Fwt/mut values greater than 1.0, and mutations that resultin significantly deleterious effects (Fwt/mut >10) are shown in boldtext. Several mutations were not observed amongst the target selectionand only a lower limit could be defined for its wt/mut ratio; therefore,the Fwt/mut value was indicated as a greater sign.

FIGS. 17A and 17B describe the results of the G6 and G6-23 light chainshotgun scan. The effect of each mutation (FIGS. 14A and B) on the lightchain CDR residues of (A) G6 and (B) G6-23 was evaluated by calculatingwt/mut ratios from sequences of functional clones, either from thealanine-scan libraries (m1, m2, and m3) or the homolog-scan libraries(m4 and m5), isolated after binding selection to either hVEGF (targetselection) or an anti-gD tag antibody (display selection). To provide aquantitative estimate of the effect of each mutation on the bindingaffinity of G6 and G6-23 for hVEGF, the function ratio (Fwt/mut) at eachmutation site was derived from dividing the wt/mut ratio from targetselection by that from display selection. Deleterious effects areindicated by Fwt/mut values greater than 1.0, and mutations that resultin significantly deleterious effects (Fwt/mut >10) are shown in boldtext.

FIG. 18 is a comparison of relative binding activities for FabG6 andG6-23 point mutants to hVEGF with function values (Fwt/mut) from shotgunscanning. The relative binding activities of each mutant protein tohVEGF were evaluated with IC50,mut/IC50,wt ratio, a measure of the foldreduction in hVEGF binding activity due to each point mutation. TheIC50, wt values for G6 and G6-23 are 2.5 nM and 20 pM respectively. Theratio without showing the standard error is estimated the error is ±5%.The ratios of significantly deleterious mutations could not be preciselyquantitated because no inhibition was observed at the highestconcentration of hVEGF (1 uM and 0.1 uM for G6 and G6-23 respectively)used in the assay and were only shown as a lower limit (>400 and >5000for G6 and G6-23 mutants respectively) for fold reduction in hVEGFbinding. The function values (Fwt/mut) of shotgun scanning were fromFIGS. 16 and 17. DDGmut-wt values for both mutagenesis scanning werecalculated using the equation as indicated in the legend of FIGS. 22Aand B.

FIG. 19 describes the relative binding affinities as measured by phageELISAs for phage-derived hVEGF₁₋₁₀₉ single alanine mutant to binddifferent versions of anti-hVEGF antibodies or hVEGF receptors (G6,G6-23, and B20-4 Fabs, monoclonal antibody A4.6.1 and receptors Flt-1(1-3), Flt-1_(D2) and KDR-Ig, respectively). The effects ofphage-derived single alanine mutants of hVEGF on the binding affinity ofanti-hVEGF antibody and hVEGF receptor were assessed as the relativeIC50 values, which are calculated as IC_(50,ala)/ID_(50,wt) from phageELISAs. A number greater than 1.0 indicated reduction in bindingaffinity and any variant with significant relative IC50 values greaterthan 10 was highlighted as bold text. The IC_(50,wt) values for G6 andG6-23 are 2.5 nM and 20 pM respectively. The ratio without showing thestandard error is estimated that the average error is +/−5%. Becausephage ELISAs required substantial binding of the mutant phagemid to itsprotein target to generate a measurable signal, non-binders (NB) couldnot be precisely quantitated but may be interpreted to have a greatlyreduced binding affinity (greater than 1000 IC50 value). Asterick (*)indicated the alanine scanning data on hVEGF for Fab-12 and hVEGFreceptors were from Muller et al., (1997) PNAS USA 94: 7192-7197 and L1et al., (2000) Cancer Res. 57:4593-4599.

FIGS. 20A and 20B describe results from the FabG6 and G6-23 heavy chainshotgun scan. Fwt/mut values measured the effects of FabG6 and G6-23heavy chain CDRs alanine (black bars) or homolog (white bars)substitutions on binding affinity for hVEGF. Shotgun scanning data for(A) FabG6 heavy chain were from FIG. 16A, and for (B) FabG6-23 heavychain were from FIG. 16B.

FIGS. 21A and 21B describe results from the FabG6 and G6-23 light chainshotgun scan. Fwt/mut values measured the effects of FabG6 and G6-23light chain CDRs alanine (black bars) or homolog (white bars)substitutions on binding affinity for hVEGF. Shotgun scanning data for(A) FabG6 light chain were from FIG. 17A, and for (B) FabG6-23 lightchain were from FIG. 17B.

FIGS. 22A and 22B report the DDGAla-wt values for the FabG6 and G6-23shotgun scan. DDGAla-wt values measuring the effects of (A) FabG6 and(B) FabG6-23 CDRs alanine substitutions on binding affinity for hVEGFwere calculated using the biophysical equation(DDGAla-wt=RTln(Ka,wt/Ka,Ala)=RTln(Fwt/Ala)) as previously described(Weiss et al., (2000) PNAS USA 97:8950-8954) and Fwt/Ala values werefrom FIGS. 16 and 17.

FIG. 23 describes the results of sensograms for injection of 100 nM Fabat 25° C. over hVEGF immobilized on the BIAcore chip. It demonstratedthe on-rate improvement for G6 variant with alanine substitution atposition 58 of CDR-H2. Additional on-rate improvement also can beobserved for this variant with valine substitution at position 51 ofCDR-H2. This figure was generated using GraphPad Prism 4.0 versionsoftware (http://www.graphpad.com).

FIG. 24 shows the amino acid sequences of the G6 Fab light chain and theG6 Fab heavy chain, respectively (SEQ ID NO. 28 and 31). The underlinesindicate residues in the CDR1-LC, CDR2-LC and CDR3-LC or CDR1-HC,CDR2-HC or CDR3-HC according to the Kabat numbering system.

FIG. 25 shows the amino acid sequence of the G6-23 Fab light chain andof the G6-23 Fab heavy chain as well as the amino acid sequences of theG6-31 and G6-8 Fab light chains (SEQ ID NO. 30-33). The underlinesindicate the position of the CDRs according to the Kabat numberingsystem.

FIG. 26 shows the amino acid sequences of the G6-23.1 and G6-23.2 Fablight and heavy chains (SEQ ID NO. 34-36). The underlines indicate theposition of the CDRs according to the Kabat numbering system.

FIG. 27 shows the amino acid sequence of the B20 Fab light and heavychains (SEQ ID NO. 37-38). The underlines indicate the position of theCDRs according to the Kabat numbering system.

FIG. 28 shows a summary of high affinity binders of VEGF derived from aB20-based library (SEQ ID NO. 85-140).

FIG. 29 shows the amino acid sequence of the B20-4 and B20-4.1 Fab lightand heavy chains (SEQ ID NO. 39-41).

FIGS. 30A and 30B show the affinity improvement of the anti-mVEGF Fab G6by light chain randomization. (A) the sensograms for injection of 500 nMFab at 37° C. over mVEGF immobilized BIAcore chip demonstrate the offrate and on rate improvements; (B) the observed rate (K_(obs), s⁻¹) ofcomplex formation between G6 and variant Fabs and mVEGF as the rate ofthe decrease of fluorescence intensity was plotted against theconcentrations of mVEGF (nM) used and the slope based on thepseudo-first-order analysis was the on rate (×10⁹ M⁻¹ s⁻¹).

FIGS. 31A and 31B show that G6 can effectively block both hVEGF andmVEGF binding to Flt-1 receptor. Fab-12 and Y0317 were used as controls.

FIG. 32 shows on-rates, off-rates, Kd values and IC50 values for Fabprotein-human VEGF or Fab protein-mouse VEGF interactions at 25° C. and37° C. using surface plasmon resonance (SPR) methods with BIAcore orsolution binding assays with competition ELISA or fluorescence quenchingwith Fab-12 and Y0317 as comparison. “NB” indicates non-binder. “ND”indicates not determined

FIG. 33A-33E shows the inhibition of HM7 tumor growth in nude mice afteradministration with the G6 antibody, the G6-31 antibody, the Y0317antibody and the Avastin™ antibody at (A) 0.1 mg/kg dose twice weekly,(B) 0.25 mg/kg dose twice weekly; (C) 0.5 mg/kg dose twice weekly; (D) 2mg/kg twice weekly and (E) 5 mg/kg dose twice weekly. An anti-ragweedantibody was used as a control.

FIG. 34 shows a summary of various G6 (A) light chain and (B) heavychain variants based on sequence and IC50 analysis (SEQ ID NO. 361-488).The wild type IC50 reflects the mean of the values obtained in thedifferent experiments. Varied amino acid positions are shown in bold.Residues differing from wild type G6 are highlighted. The residues aredenoted by the single-letter amino acid code and a number denoting itsposition according to the scheme of Kabat et al., 1987.

FIG. 35 shows a summary of various G6-23 (A) light chain and (B) heavychain variants based on sequence and IC50 analysis (SEQ ID NO. 141-272).The wild type IC50 reflects the mean of the values obtained in thedifferent experiments. Varied amino acid positions are shown in bold.Residues differing from wild type G6 are highlighted. The residues aredenoted by the single-letter amino acid code and a number denoting itsposition according to the scheme of Kabat et al., 1987.

FIG. 36 shows portions of the amino acid sequences of YADS-1, YADS-2 andYADS-3 antibodies and other clones (SEQ ID NO. 489-560). Represented aresequences of three hVEGF binders selected from the YADS-II library.Residues that were not randomized in the library are grey shaded.

FIG. 37 shows amino acid sequences of a group of YADS series antibodies(SEQ ID NO. 273-332). Represented are sequences of unique clonesobtained from the sorting of library YADS-A against hVEGF. Residues thatwere not randomized in the library are grey shaded.

FIG. 38 shows amino acid sequences of a group of YADS series antibodies(SEQ ID NO. 333-360). Represented are sequences of unique clonesobtained from the sorting of library YADS-B against hVEGF. Residues thatwere not randomized in the library are grey shaded.

FIG. 39 shows the Fab sequences of YADS2 and YADS3 antibodies (SEQ IDNO. 21-24). Bolded portions indicate variable region resides. Underlinedportions indicate approximate residues of CDR-L1, CDR-L2, CDR-L3,CDR-H1, CDR-H2 or CDR-H3.

FIG. 40 shows NNK variants of the YADS2 antibody (SEQ ID NO. 723-794).“x” indicates a STOP codon or an unreadable sequence.

FIG. 41 shows amino acid sequences of a group of YS series antibodies(SEQ ID NO. 795-914). They represent sequences of binders obtained fromselection of library YS-A and YS-B.

FIG. 42 shows CDR sequences from anti-VEGF antibodies of the YSlibraries (SEQ ID NO. 561-722). Clones 1-52 were selected from libraryB, while clones 53-63 were selected from library A. From top to bottom,columns 1-5 of CDR-L3 refer to positions 91, 92, 93, 94 and 96,respectively; columns 1-5 of CDR-H1 refer to positions 28, 30, 31, 32,and 33; columns 1-6 of CDR-H2 refer to positions 50, 52, 53, 54, 56 and58; and columns 1-15 of CDR-H3 refer to positions 95, 96, 97, 98, 99,100 and 100a-i according to Kabat numbering.

FIG. 43 shows the sequence of the YS1 Fab. Portions in bold indicateresidues in the variable region (SEQ ID NO. 42-43).

FIG. 44 shows that the growth of HUVEC cells induced by VEGF isinhibited by YADS2 Fab.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. A “G6 series polypeptide” according tothis invention is a polypeptide, including an antibody according to thisinvention, that is derived from a sequence of a G6 antibody orG6-derived antibody according to any one of FIGS. 7, 24-26 and 34-35 andbinds to human VEGF with a desired affinity according to this invention(e.g., 10 nm or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 500 pMor less for the Fab version of the antibody at 25° C.). A “B20 seriespolypeptide” according to this invention is a polypeptide, including anantibody according to this invention, that is derived from a sequence ofthe B20 antibody or a B20-derived antibody according to any one of FIGS.27-29 and binds to VEGF with a desired affinity according to thisinvention. A “YADS series polypeptide” or “YADS polypeptide” accordingto this invention is a polypeptide, including an antibody according tothis invention, that is derived from a sequence of the YADS antibodyaccording to any one of FIGS. 36-40 and binds to VEGF with a desiredaffinity according to this invention. A “YADS-2 series polypeptide” or“YADS2 polypeptide” according to this invention is a polypeptide,including an antibody according to this invention, that is derived froma sequence of the YADS2 antibody according to FIG. 36 or FIG. 39 andbinds to VEGF with a desired affinity according to this invention. A“YADS-3 series polypeptide” or “YADS3 polypeptide” according to thisinvention is a polypeptide, including an antibody according to thisinvention, that is derived from a sequence of the YADS3 antibodyaccording to FIG. 36 or FIG. 39 and binds to VEGF with a desiredaffinity according to this invention. “YS series polypeptide” or “YSpolypeptide” according to this invention is a polypeptide, including anantibody according to this invention, that is derived from a sequence ofa YS antibody according to FIG. 41-43 and binds to VEGF with a desiredaffinity according to this invention. According to one preferredembodiment, the G series polypeptide, B20 series polypeptide, the YADSseries polypeptide, the YADS2 series polypeptide, the YADS3 seriespolypeptide or the YS series polypeptide binds to human and a non-humanmammalian VEGF with a Kd value that is within 10 fold of each other.According to one embodiment, the kD values for those antibodies bindingto human VEGF and a mouse VEGF are 10 nM or less. In another embodiment,the antibodies bind to human VEGF and mouse VEGF with Kd values of 2 nMor less. In another embodiment, the antibodies bind to human VEGF withKd values of 1 nM or less. The affinities of such G6 series, B20 series,YADS and YS series polypeptides for VEGF can be improved by methods,e.g., such as the phage display techniques taught herein.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2,and CDR3), and Framework Regions (FRs). V_(H) refers to the variabledomain of the heavy chain. V_(L) refers to the variable domain of thelight chain. According to the methods used in this invention, the aminoacid positions assigned to CDRs and FRs may be defined according toKabat (Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987 and 1991)). Amino acidnumbering of antibodies or antigen binding fragments is also accordingto that of Kabat.

As used herein, the term “Complementarity Determining Regions” (CDRs;i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop. For example, the CDRH1 of the heavy chain of antibody 4D5 includesamino acids 26 to 35.

“Framework regions” (hereinafter FR) are those variable domain residuesother than the CDR residues. Each variable domain typically has four FRsidentified as FR1, FR2, FR3 and FR4. If the CDRs are defined accordingto Kabat, the light chain FR residues are positioned at about residues1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and theheavy chain FR residues are positioned about at residues 1-30 (HCFR1),36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chainresidues. If the CDRs comprise amino acid residues from hypervariableloops, the light chain FR residues are positioned about at residues 1-25(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the lightchain and the heavy chain FR residues are positioned about at residues1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in theheavy chain residues. In some instances, when the CDR comprises aminoacids from both a CDR as defined by Kabat and those of a hypervariableloop, the FR residues will be adjusted accordingly. For example, whenCDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are atpositions 1-25 and the FR2 residues are at positions 36-49.

As used herein, “codon set” refers to a set of different nucleotidetriplet sequences used to encode desired variant amino acids. A set ofoligonucleotides can be synthesized, for example, by solid phasesynthesis, including sequences that represent all possible combinationsof nucleotide triplets provided by the codon set and that will encodethe desired group of amino acids. A standard form of codon designationis that of the IUB code, which is known in the art and described herein.A codon set typically is represented by 3 capital letters in italics,eg. NNK, NNS, XYZ, DVK and the like. A “non-random codon set”, as usedherein, thus refers to a codon set that encodes select amino acids thatfulfill partially, preferably completely, the criteria for amino acidselection as described herein. Synthesis of oligonucleotides withselected nucleotide “degeneracy” at certain positions is well known inthat art, for example the TRIM approach (Knappek et al.; J. Mol. Biol.(1999), 296:57-86); Garrard & Henner, Gene (1993), 128:103). Such setsof oligonucleotides having certain codon sets can be synthesized usingcommercial nucleic acid synthesizers (available from, for example,Applied Biosystems, Foster City, Calif.), or can be obtainedcommercially (for example, from Life Technologies, Rockville, Md.).Therefore, a set of oligonucleotides synthesized having a particularcodon set will typically include a plurality of oligonucleotides withdifferent sequences, the differences established by the codon set withinthe overall sequence. Oligonucleotides, as used according to theinvention, have sequences that allow for hybridization to a variabledomain nucleic acid template and also can, but does not necessarily,include restriction enzyme sites useful for, for example, cloningpurposes.

An “Fv” fragment is an antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight association,which can be covalent in nature, for example in scFv. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′)₂ antibody fragments comprise a pair of Fab fragmentswhich are generally covalently linked near their carboxy termini byhinge cysteines between them. Other chemical couplings of antibodyfragments are also known in the art.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The expression “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen (i.e.has a binding affinity (K_(d)) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ M and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second nonhuman mammalian species which is at least about50 fold, or at least about 500 fold, or at least about 1000 fold, weakerthan its binding affinity for the human antigen. The species-dependentantibody can be any of the various types of antibodies as defined above,but preferably is a humanized or human antibody.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Ina preferred embodiment, the antibody mutant will have an amino acidsequence having at least 75% amino acid sequence identity or similaritywith the amino acid sequence of either the heavy or light chain variabledomain of the species-dependent antibody, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e same residue) or similar(i.e. amino acid residue from the same group based on common side-chainproperties, see below) with the species-dependent antibody residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence outside of the variable domain shall be construed asaffecting sequence identity or similarity.

To increase the half-life of the antibodies or polypeptide containingthe amino acid sequences of this invention, one can attach a salvagereceptor binding epitope to the antibody (especially an antibodyfragment), as described, e.g., in U.S. Pat. No. 5,739,277. For example,a nucleic acid molecule encoding the salvage receptor binding epitopecan be linked in frame to a nucleic acid encoding a polypeptide sequenceof this invention so that the fusion protein expressed by the engineerednucleic acid molecule comprises the salvage receptor binding epitope anda polypeptide sequence of this invention. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule (e.g.,Ghetie, V et al., (2000) Ann. Rev. Immunol. 18:739-766, Table 1).Antibodies with substitutions in an Fc region thereof and increasedserum half-lives are also described in WO00/42072 (Presta, L.), WO02/060919; Shields, R. L., et al., (2001) JBC 276(9):6591-6604; Hinton,P. R., (2004) JBC 279(8):6213-6216). In another embodiment, the serumhalf-life can also be increased, for example, by attaching otherpolypeptide sequences. For example, antibodies of this invention orother polypeptide containing the amino acid sequences of this inventioncan be attached to serum albumin or a portion of serum albumin thatbinds to the FcRn receptor or a serum albumin binding peptide so thatserum albumin binds to the antibody or polypeptide, e.g., suchpolypeptide sequences are disclosed in WO01/45746. In one preferredembodiment, the serum albumin peptide to be attached comprises an aminoacid sequence of DICLPRWGCLW. In another embodiment, the half-life of aFab according to this invention is increased by these methods. See also,Dennis, M. S., et al., (2002) JBC 277(38):35035-35043 for serum albuminbinding peptide sequences.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promote angiogenesis, endothelialcell growth, stability of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, P1GF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, Del-1,fibroblast growth factors: acidic (aFGF) and basic (bFGF), Follistatin,Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,Placental growth factor, Platelet-derived endothelial cell growth factor(PD-ECGF), Platelet-derived growth factor, especially PDGF-BB orPDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin, Transforminggrowth factor-alpha (TGF-alpha), Transforming growth factor-beta(TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Vascularendothelial growth factor (VEGF)/vascular permeability factor (VPF),etc. It would also include factors that accelerate wound healing, suchas growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermalgrowth factor (EGF), CTGF and members of its family, and TGF-alpha andTGF-beta. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol.,53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003);Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini etal., Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listing knownangiogenic factors); and, Sato Int. J. Clin. Oncol., 8:200-206 (2003).

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as defined above,e.g., antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptoror Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec™ (ImatinibMesylate). Anti-angiogenesis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar,Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenictherapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003)(e.g., Table 2 listing known antiangiogenic factors); and, Sato Int. J.Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 lists anti-angiogenicagents used in clinical trials).

The “Kd” or “Kd value” according to this invention is in one preferredembodiment measured by a radiolabeled VEGF binding assay (RIA) performedwith the Fab version of the antibody and a VEGF molecule as described bythe following assay that measures solution binding affinity of Fabs forVEGF by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeledVEGF(109) in the presence of a titration series of unlabeled VEGF, thencapturing bound VEGF with an anti-Fab antibody-coated plate (Chen, etal., (1999) J. Mol Biol 293:865-881). To establish conditions for theassay, microtiter plates (Dynex) are coated overnight with 5 ug/ml of acapturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBSfor two to five hours at room temperature (approximately 23° C.). In anon-adsorbant plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]VEGF(109) aremixed with serial dilutions of a Fab of interest, e.g., Fab-12 (Prestaet al., (1997) Cancer Res. 57:4593-4599). The Fab of interest is thenincubated overnight; however, the incubation may continue for 65 hoursto insure that equilibrium is reached. Thereafter, the mixtures aretransferred to the capture plate for incubation at room temperature forone hour. The solution is then removed and the plate washed eight timeswith 0.1% Tween-20 in PBS. When the plates had dried, 150 ul/well ofscintillant (MicroScint-20; Packard) is added, and the plates arecounted on a Topcount gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.According to another embodiment the Kd or Kd value is measured by usingsurface plasmon resonance assays using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized hVEGF (8-109) CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Human VEGF is diluted with 10 mM sodiumacetate, pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rateof 5 ul/minute to achieve approximately 10 response units (RU) ofcoupled protein. Following the injection of human VEGF, 1M ethanolamineis injected to block unreacted groups. For kinetics measurements,two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBSwith 0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25ul/min Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgram. The equilibrium dissociationconstant (Kd) was calculated as the ratio k_(off)/k_(on). See, e.g.,Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds10⁶M⁻¹ S⁻¹ by the surface plasmon resonance assay above, then theon-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-VEGF antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of human VEGF short form (8-109)or mouse VEGF as measured in a spectrometer, such as a stop-flowequipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Amincospectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention is preferably determined with same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized hVEGF (8-109) CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Human VEGF is diluted with 10 mM sodiumacetate, pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rateof 5 ul/minute to achieve approximately 10 response units (RU) ofcoupled protein. Following the injection of 1M ethanolamine to blockunreacted groups. For kinetics measurements, two-fold serial dilutionsof Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20(PBST) at 25° C. at a flow rate of approximately 25 ul/min Associationrates (k_(on)) and dissociation rates (k_(off)) are calculated using asimple one-to-one Langmuir binding model (BIAcore Evaluation Softwareversion 3.2) by simultaneously fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. MolBiol 293:865-881. However, if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate is preferablydetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-VEGFantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of human short form (8-109) or mouse VEGF as measured ina a spectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stirred cuvette.

A “functional epitope” according to this invention refers to amino acidresidues of an antigen that contribute energetically to the binding ofan antibody. Mutation of any one of the energetically contributingresidues of the antigen (for example, mutation of wild-type VEGF byalanine or homolog mutation) will disrupt the binding of the antibodysuch that the relative affinity ratio (IC50 mutant VEGF/IC50 wild-typeVEGF) of the antibody will be greater than 5 (see Example 2). In apreferred embodiment, the relative affinity ratio is determined by asolution binding phage displaying ELISA. Briefly, 96-well Maxisorpimmunoplates (NUNC) are coated overnight at 4° C. with an Fab form ofthe antibody to be tested at a concentration of 2 μg/ml in PBS, andblocked with PBS, 0.5% BSA, and 0.05% Tween20 (PBT) for 2 h at roomtemperature. Serial dilutions of phage displaying hVEGF alanine pointmutants (residues 8-109 form) or wild type hVEGF (8-109) in PBT arefirst incubated on the Fab-coated plates for 15 min at room temperature,and the plates are washed with PBS, 0.05% Tween20 (PBST). The boundphage is detected with an anti-M13 monoclonal antibody horseradishperoxidase (Amersham Pharmacia) conjugate diluted 1:5000 in PBT,developed with 3,3′,5,5′-tetramethylbenzidine (TMB, Kirkegaard & PerryLabs, Gaithersburg, Md.) substrate for approximately 5 min, quenchedwith 1.0 M H₃PO₄, and read spectrophotometrically at 450 nm. The ratioof IC50 values (IC50,ala/IC50,wt) represents the fold of reduction inbinding affinity (the relative binding affinity).

For competitive binding assays, Maxisorb plates are coated and blockedas above, and serial threefold dilutions of unlabeled VEGF(109) are madein PBS/Tween buffer in a Nunc plate. [¹²⁵I]VEGF(109) is added, followedby addition of a fixed concentration of the Fab of interest. The finalconcentrations of the Fab of interest are 100 pM and 10 pM,respectively. After incubation (as above), bound VEGF is captured andquantified as described above. The binding data is analyzed using acomputer program to perform Scatchard analysis (P. Munson et al., Anal.Biochem. (1980) 107:220-239) for determination of the dissociationbinding constants, K_(d).

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (for example, polypeptides of the invention), or thenucleic acids that encode these sequences, the sequences being differentin the combination of variant amino acids that are introduced into thesesequences according to the methods of the invention.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target antigen with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct.Biol., 3:355-362 (1992), and references cited therein. In a monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells, Methods: A companion to Methods inEnzymology, 3:205-216 (1991).

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., ColE1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13, fl, fd, Pf3 phage or aderivative thereof, or a lambdoid phage, such as lambda, 21, phi80,phi81, 82, 424, 434, etc., or a derivative thereof.

As used herein, “solvent accessible position” refers to a position of anamino acid residue in the variable regions of the heavy and light chainsof a source antibody or antigen binding fragment that is determined,based on structure, ensemble of structures and/or modeled structure ofthe antibody or antigen binding fragment, as potentially available forsolvent access and/or contact with a molecule, such as anantibody-specific antigen. These positions are typically found in theCDRs and on the exterior of the protein. The solvent accessiblepositions of an antibody or antigen binding fragment, as defined herein,can be determined using any of a number of algorithms known in the art.Preferably, solvent accessible positions are determined usingcoordinates from a 3-dimensional model of an antibody, preferably usinga computer program such as the InsightII program (Accelrys, San Diego,Calif.). Solvent accessible positions can also be determined usingalgorithms known in the art (e.g., Lee and Richards, J. Mol. Biol. 55,379 (1971) and Connolly, J. Appl. Cryst. 16, 548 (1983)). Determinationof solvent accessible positions can be performed using software suitablefor protein modeling and 3-dimensional structural information obtainedfrom an antibody. Software that can be utilized for these purposesincludes SYBYL Biopolymer Module software (Tripos Associates). Generallyand preferably, where an algorithm (program) requires a user input sizeparameter, the “size” of a probe which is used in the calculation is setat about 1.4 Angstrom or smaller in radius. In addition, determinationof solvent accessible regions and area methods using software forpersonal computers has been described by Pacios ((1994)“ARVOMOL/CONTOUR: molecular surface areas and volumes on PersonalComputers.” Comput. Chem. 18(4): 377-386; and (1995). “Variations ofSurface Areas and Volumes in Distinct Molecular Surfaces ofBiomolecules.” J. Mol. Model. 1: 46-53.)

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment with theantibody. For example, mammals who suffer from or need prophylaxisagainst abnormal angiogenesis (excessive, inappropriate or uncontrolledangiogenesis) or vascular permeability. This includes chronic and acutedisorders or diseases including those pathological conditions whichpredispose the mammal to the disorder in question. Non-limiting examplesof disorders to be treated herein include malignant and benign tumors;non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, angiogenic and immunologicdisorders.

The term abnormal angiogenesis occurs when new blood vessels either growexcessively, insufficiently or inappropriately (e.g., the location,timing or onset of the angiogenesis being undesired from a medicalstandpoint) in a diseased state or such that it causes a diseased state.Excessive, inappropriate or uncontrolled angiogenesis occurs when thereis new blood vessel growth that contributes to the worsening of thediseased state or causes a diseased state, such as in cancer, especiallyvascularized solid tumors and metastatic tumors (including colon, lungcancer (especially small-cell lung cancer), or prostate cancer),diseases caused by ocular neovascularisation, especially diabeticblindness, retinopathies, primarily diabetic retinopathy or age-inducedmacular degeneration and rubeosis; psoriasis, psoriatic arthritis,haemangioblastoma such as haemangioma; inflammatory renal diseases, suchas glomerulonephritis, especially mesangioproliferativeglomerulonephritis, haemolytic uremic syndrome, diabetic nephropathy orhypertensive nephrosclerosis; various imflammatory diseases, such asarthritis, especially rheumatoid arthritis, inflammatory bowel disease,psorsasis, sarcoidosis, arterial arteriosclerosis and diseases occurringafter transplants, endometriosis or chronic asthma and more than 70other conditions. The new blood vessels can feed the diseased tissues,destroy normal tissues, and in the case of cancer, the new vessels canallow tumor cells to escape into the circulation and lodge in otherorgans (tumor metastases). Insufficient angiogenesis occurs when thereis inadequate blood vessel growth that contributes to the worsening of adiseased state, e.g., in diseases such as coronary artery disease,stroke, and delayed wound healing. Further, ulcers, strokes, and heartattacks can result from the absence of angiogenesis that is normallyrequired for natural healing. The present invention contemplatestreating those patients that are at risk of developing theabove-mentioned illnesses.

Other patients that are candidates for receiving the antibodies orpolypeptides of this invention have, or are at risk for developing,abnormal proliferation of fibrovascular tissue, acne rosacea, acquiredimmune deficiency syndrome, artery occlusion, atopic keratitis,bacterial ulcers, Bechets disease, blood borne tumors, carotidobstructive disease, choroidal neovascularization, chronic inflammation,chronic retinal detachment, chronic uveitis, chronic vitritis, contactlens overwear, corneal graft rejection, corneal neovascularization,corneal graft neovascularization, Crohn's disease, Eales disease,epidemic keratoconjunctivitis, fungal ulcers, Herpes simplex infections,Herpes zoster infections, hyperviscosity syndromes, Kaposi's sarcoma,leukemia, lipid degeneration, Lyme's disease, marginal keratolysis,Mooren ulcer, Mycobacteria infections other than leprosy, myopia, ocularneovascular disease, optic pits, Osler-Weber syndrome(Osler-Weber-Rendu), osteoarthritis, Pagets disease, pars planitis,pemphigoid, phylectenulosis, polyarteritis, post-laser complications,protozoan infections, pseudoxanthoma elasticum, pterygium keratitissicca, radial keratotomy, retinal neovascularization, retinopathy ofprematurity, retrolental fibroplasias, sarcoid, scleritis, sickle cellanemia, Sjögren's syndrome, solid tumors, Stargarts disease,Stevens-Johnson disease, superior limbic keratitis, syphilis, systemiclupus, Terrien's marginal degeneration, toxoplasmosis, trauma, tumors ofEwing sarcoma, tumors of neuroblastoma, tumors of osteosarcoma, tumorsof retinoblastoma, tumors of rhabdomyosarcoma, ulcerative colitis, veinocclusion, Vitamin A deficiency and Wegeners sarcoidosis, undesiredangiogenesis associated with diabetes, parasitic diseases, abnormalwound healing, hypertrophy following surgery, injury or trauma,inhibition of hair growth, inhibition of ovulation and corpus luteumformation, inhibition of implantation and inhibition of embryodevelopment in the uterus.

Anti-angiogenesis therapies are useful in the general treatment of graftrejection, lung inflammation, nephrotic syndrome, preeclampsia,pericardial effusion, such as that associated with pericarditis, andpleural effusion, diseases and disorders characterized by undesirablevascular permeability, e.g., edema associated with brain tumors, ascitesassociated with malignancies, Meigs' syndrome, lung inflammation,nephrotic syndrome, pericardial effusion, pleural effusion, permeabilityassociated with cardiovascular diseases such as the condtion followingmyocardial infarctions and strokes and the like.

Other angiogenesis-dependent diseases according to this inventioninclude angiofibroma (abnormal blood of vessels which are prone tobleeding), neovascular glaucoma (growth of blood vessels in the eye),arteriovenous malformations (abnormal communication between arteries andveins), nonunion fractures (fractures that will not heal),atherosclerotic plaques (hardening of the arteries), pyogenic granuloma(common skin lesion composed of blood vessels), scleroderma (a form ofconnective tissue disease), hemangioma (tumor composed of bloodvessels), trachoma (leading cause of blindness in the third world),hemophilic joints, vascular adhesions and hypertrophic scars (abnormalscar formation).

The term “VEGF” or “VEGF” as used herein refers to the 165-amino acidhuman vascular endothelial cell growth factor and related 121-, 189-,and 206 amino acid human vascular endothelial cell growth factors, asdescribed by Leung et al. Science, 246:1306 (1989), and Houck et al.Mol. Endocrin., 5:1806 (1991), together with the naturally occurringallelic and processed forms thereof. The term “VEGF” also refers toVEGFs from non-human species such as mouse, rat or primate. Sometimesthe VEGF from a specific species are indicated by terms such as hVEGFfor human VEGF, mVEGF for murine VEGF, and etc. The term “VEGF” is alsoused to refer to truncated forms of the polypeptide comprising aminoacids 8 to 109 or 1 to 109 of the 165-amino acid human vascularendothelial cell growth factor. Reference to any such forms of VEGF maybe identified in the present application, e.g., by “VEGF (8-109),” “VEGF(1-109)” or “VEGF₁₆₅.” The amino acid positions for a “truncated” nativeVEGF are numbered as indicated in the native VEGF sequence. For example,amino acid position 17 (methionine) in truncated native VEGF is alsoposition 17 (methionine) in native VEGF. The truncated native VEGF hasbinding affinity for the KDR and Flt-1 receptors comparable to nativeVEGF.

The term “VEGF variant” as used herein refers to a VEGF polypeptidewhich includes one or more amino acid mutations in the native VEGFsequence. Optionally, the one or more amino acid mutations include aminoacid substitution(s). For purposes of shorthand designation of VEGFvariants described herein, it is noted that numbers refer to the aminoacid residue position along the amino acid sequence of the putativenative VEGF (provided in Leung et al., supra and Houck et al., supra.).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals,nonhuman primates, and zoo, sports, or pet animals, such as dogs,horses, cats, cows, etc.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent.” Examples of therapeutic agents (anti-cancer agents)include, but are not limited to, e.g., chemotherapeutic agents, growthinhibitory agents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, andother-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g.,a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib(Tarceva™), platelet derived growth factor inhibitors (e.g., Gleevec™(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons,cytokines, antagonists (e.g., neutralizing antibodies) that bind to oneor more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, B1yS,APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive andorganic chemical agents, etc. Combinations thereof are also included inthe invention.

The term “epitope tagged” when used herein refers to an antibody mutantfused to an “epitope tag”. The epitope tag polypeptide has enoughresidues to provide an epitope against which an antibody thereagainstcan be made, yet is short enough such that it does not interfere withactivity of the antibody mutant. The epitope tag preferably also isfairly unique so that the antibody thereagainst does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least 6 amino acid residues, and usually between about 8-50amino acid residues (preferably between about 9-30 residues). Examplesinclude the flu HA tag polypeptide and its antibody 12CA5 (Field et al.Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7,6E10, G4, B7 and 9E10 antibodies thereagainst (Evan et al., Mol. Cell.Biol. 5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody (Paborsky et al., Protein Engineering3(6):547-553 (1990)). In certain embodiments, the epitope tag is a“salvage receptor binding epitope”.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include achemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomega1I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenishers such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elflornithine; elliptinium acetate; an epothilone;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide;procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene,Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton,N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticleformulation of paclitaxel (American Pharmaceutical Partners, Schaumberg,Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France);chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,erlotinib (Tarceva™)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation, such as, for example, PKC-alpha, Raf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No.4,675,187), and pharmaceutically acceptable salts, acids or derivativesof any of the above.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

For the treatment of rheumatoid arthritis, the patient can be treatedwith an antibody of the invention in conjunction with any one or more ofthe following drugs: DMARDS (disease-modifying anti-rheumatic drugs(e.g., methotrexate), NSAI or NSAID (non-steroidal anti-inflammatorydrugs), HUMIRA™ (adalimumab; Abbott Laboratories), ARAVA® (leflunomide),REMICADE® (infliximab; Centocor Inc., of Malvern, Pa.), ENBREL™(etanercept; Immunex, WA), COX-2 inhibitors. DMARDs commonly used in RAare hydroxycloroquine, sulfasalazine, methotrexate, leflunomide,etanercept, infliximab, azathioprine, D-penicillamine, Gold (oral), Gold(intramuscular), minocycline, cyclosporine, Staphylococcal protein Aimmunoadsorption. Adalimumab is a human monoclonal antibody that bindsto TNFα. Infliximab is a chimeric monoclonal antibody that binds toTNFα. Etanercept is an “immunoadhesin” fusion protein consisting of theextracellular ligand binding portion of the human 75 kD (p75) tumornecrosis factor receptor (TNFR) linked to the Fc portion of a humanIgG1. For conventional treatment of RA, see, e.g., “Guidelines for themanagement of rheumatoid arthritis” Arthritis & Rheumatism 46(2):328-346 (February, 2002). In a specific embodiment, the RA patient istreated with a CD20 antibody of the invention in conjunction withmethotrexate (MTX). An exemplary dosage of MTX is about 7.5-25 mg/kg/wk.MTX can be administered orally and subcutaneously.

For the treatment of ankylosing spondylitis, psoriatic arthritis andCrohn's disease, the patient can be treated with an antibody of theinvention in conjunction with, for example, Remicade® (infliximab; fromCentocor Inc., of Malvern, Pa.), ENBREL (etanercept; Immunex, WA).

For treatments for SLE, the patient can be treated with an antibody ofthe invention in conjunction with, for example, high-dosecorticosteroids and/or cyclophosphamide (HDCC).

For the treatment of psoriasis, patients can be administered an antibodyof this invention in conjunction with topical treatments, such astopical steroids, anthralin, calcipotriene, clobetasol, and tazarotene,or with methotrexate, retinoids, cyclosporine, PUVA and UVB therapies.In one embodiment, the psoriasis patient is treated with the antibodysequentially or concurrently with cyclosporine.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

II. Modes for Carrying out the Invention

Synthetic High-Affinity Anti-VEGF Antibodies

The invention herein provides novel anti-VEGF antibodies with highbinding affinity to VEGF. Exemplary methods for generating antibodiesare described in more detail in the following sections.

The novel anti-VEGF antibodies are selected using the VEGF antigenderived from a first mammalian species. Preferably the antigen is humanVEGF (hVEGF). However, VEGFs from other species such as murine VEGF(mVEGF) can also be used as the first target antigen. The VEGF antigensfrom various mammalian species may be isolated from natural sources. Inother embodiments, the antigen is produced recombinantly or made usingother synthetic methods known in the art.

The antibody selected will normally have a sufficiently strong bindingaffinity for the first VEGF antigen. For example, the antibody may bindhVEGF with a K_(d) value of no more than about 5 nM, preferably no morethan about 2 nM, and more preferably no more than about 500 pM. Antibodyaffinities may be determined by a surface plasmon resonance based assay(such as the BIAcore assay as described in Examples); enzyme-linkedimmunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), forexample.

Also, the antibody may be subjected to other biological activity assays,e.g., in order to evaluate its effectiveness as a therapeutic. Suchassays are known in the art and depend on the target antigen andintended use for the antibody. Examples include the HUVEC inhibitionassay (as described in the Examples below); tumor cell growth inhibitionassays (as described in WO 89/06692, for example); antibody-dependentcellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC)assays (U.S. Pat. No. 5,500,362); and agonistic activity orhematopoiesis assays (see WO 95/27062).

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270:1388-1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

Species-specificity of the novel antibodies is then determined. Thebinding affinity of the antibody for a homologue of the antigen used toselect the antibody (where the homologue is from the “second mammalianspecies”) is assessed using techniques such as those described above. Inpreferred embodiments, the second mammalian species is a nonhuman mammalto which the antibody will be administered in preclinical studies.Accordingly, the second mammalian species may be a nonhuman primate,such as rhesus, cynomolgus, baboon, chimpanzee and macaque. In otherembodiments, the second mammalian species may be a rodent (e.g., mouseor rat), cat or dog, for example.

While the preferred method of the instant invention for determiningspecies-dependence (and for evaluating antibody mutants with improvedproperties; see below) is to quantify antibody binding affinity, inother embodiments of the invention, one or more biological properties ofthe synthetic antibody and antibody variants are evaluated in additionto, or instead of, binding affinity determinations. Exemplary suchbiological assays are described above. Such assays are particularlyuseful where they provide an indication as to the therapeuticeffectiveness of the antibody. Normally, though not necessarily,antibodies which show improved properties in such assays, will also havean enhanced binding affinity. Thus, in one embodiment of the inventionwhere the assay of choice is a biological activity assay other than abinding affinity assay, the species-dependent antibody will normallyhave a “biological activity” using “material” (e.g. antigen, cell,tissue, organ or whole animal) from the second mammalian species whichis at least about 50 fold, or at least about 500 fold, or at least about1000 fold, less effective than its biological activity in acorresponding assay using reagents from the first mammalian species.

The species-dependent antibody is then altered so as to generate anantibody mutant which has a stronger binding affinity for the antigenfrom the second mammalian species, than the species-dependent antibody.The antibody mutant preferably has a binding affinity for the antigenfrom the nonhuman mammal which is at least about 10 fold stronger,preferably at least about 20 fold stronger, more preferably at leastabout 50 fold stronger, and sometimes at least about 100 fold or 200fold stronger, than the binding affinity of the species-dependentantibody for the antigen. The enhancement in binding affinity desired orrequired will depend on the initial binding affinity of thespecies-dependent antibody. Where the assay used is a biologicalactivity assay, the antibody mutant preferably has a biological activityin the assay of choice which is at least about 10 fold better,preferably at least about 20 fold better, more preferably at least about50 fold better, and sometimes at least about 100 fold or 200 foldbetter, than the biological activity of the species-dependent antibodyin that assay.

To generate the antibody mutant, one or more amino acid alterations(e.g. substitutions) are introduced in one or more of the hypervariableregions of the species-dependent antibody. Alternatively, or inaddition, one or more alterations (e.g. substitutions) of frameworkregion residues may be introduced in the species-dependent antibodywhere these result in an improvement in the binding affinity of theantibody mutant for the antigen from the second mammalian species.Examples of framework region residues to modify include those whichnon-covalently bind antigen directly (Amit et al. Science 233:747-753(1986)); interact with/effect the conformation of a CDR (Chothia et al.J. Mol. Biol. 196:901-917 (1987)); and/or participate in the V_(L)-V_(H)interface (EP 239 400B1). In certain embodiments, modification of one ormore of such framework region residues results in an enhancement of thebinding affinity of the antibody for the antigen from the secondmammalian species. For example, from about one to about five frameworkresidues may be altered in this embodiment of the invention. Sometimes,this may be sufficient to yield an antibody mutant suitable for use inpreclinical trials, even where none of the hypervariable region residueshave been altered. Normally, however, the antibody mutant will compriseadditional hypervariable region alteration(s).

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of thespecies-dependent antibody for the antigen from the second mammalianspecies is such that such randomly produced antibody mutants can bereadily screened.

One useful procedure for generating such antibody mutants is called“alanine scanning mutagenesis” (Cunningham and Wells Science244:1081-1085 (1989)). Here, one or more of the hypervariable regionresidue(s) are replaced by alanine or polyalanine residue(s) to affectthe interaction of the amino acids with the antigen from the secondmammalian species. Those hypervariable region residue(s) demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other mutations at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. The ala-mutants produced this way arescreened for their biological activity as described herein.

Another procedure for generating such antibody mutants involves affinitymaturation using phage display (Hawkins et al. J. Mol. Biol. 254:889-896(1992) and Lowman et al. Biochemistry 30(45):10832-10837 (1991)).Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutatedto generate all possible amino substitutions at each site. The antibodymutants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed mutants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed.

The invention also provides a more systematic method for identifyingamino acid residues to modify. According to this method, one identifieshypervariable region residues in the species-dependent antibody whichare involved in binding the antigen from the first mammalian species andthose hypervariable region residues involved in binding a homologue ofthat antigen from the second mammalian species. To achieve this, analanine-scan of the hypervariable region residues of thespecies-dependent antibody can be performed, with each ala-mutant beingtested for binding to the antigen from the first and second mammalianspecies. Alternatively, the X-ray crystal structures of antibody-antigencomplexes are analyzed for contacting residues as well as surroundingresidues (as described in Examples). The hypervariable region residuesinvolved in binding the antigen from the first mammalian species (e.g.human), and those involved in binding the homologue of the antigen fromthe second mammalian species (e.g. nonhuman mammal) are therebyidentified. Preferably, those residue(s) significantly involved inbinding the antigen from the second mammalian species (e.g. nonhumanmammal), but not the antigen from the first mammalian species (e.g.human), are chosen as candidates for modification. In anotherembodiment, those residue(s) significantly involved in binding theantigen from both the first and second mammalian species are selected tobe modified (see Example below). In yet a further but less preferredembodiment, those residues involved in binding the antigen from thefirst mammalian species, but not the second mammalian species, areselected for modification. Such modification can involve deletion of theresidue or insertion of one or more residues adjacent to the residue.However, normally the modification involves substitution of the residuefor another amino acid.

Normally one would start with a conservative substitution such as thoseshown below under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity (e.g. bindingaffinity), then more substantial changes, denominated “exemplarysubstitutions” in the following table, or as further described below inreference to amino acid classes, are introduced and the productsscreened.

Preferred Substitutions:

Original Exemplary Preferred Residue Substitutions Substitutions Ala (A)val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arggln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly(G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met;ala; leu phe; norleucine Leu (L) norleucine; ile; val; ile met; ala; pheLys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val;ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;phe; leu ala; norleucine

Even more substantial modifications to the antibodies' biologicalproperties are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr, asn, gln;

(3) acidic: asp, glu;

(4) basic: his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

In another embodiment, the sites selected for modification are affinitymatured using phage display (see above).

Nucleic acid molecules encoding amino acid sequence mutants are preparedby a variety of methods known in the art. These methods include, but arenot limited to, oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared mutantor a non-mutant version of the species-dependent antibody. The preferredmethod for making mutants is site directed mutagenesis (see, e.g.,Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985)).

In certain embodiments, the antibody mutant will only have a singlehypervariable region residue substituted. In other embodiments, two ormore of the hypervariable region residues of the species-dependentantibody will have been substituted, e.g. from about two to about tenhypervariable region substitutions. For example, the murinized anti-VEGFantibody variant of the example below had four hypervariable regionsubstitutions.

Ordinarily, the antibody mutant with improved biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the species-dependent antibody, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, and most preferably at least 95%. Identity or similaritywith respect to this sequence is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical (i.esame residue) or similar (i.e. amino acid residue from the same groupbased on common side-chain properties, see above) with thespecies-dependent antibody residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence outside of thevariable domain shall be construed as affecting sequence identity orsimilarity.

Following production of the antibody mutant, the biological activity ofthat molecule relative to the species-dependent antibody is determined.As noted above, this may involve determining the binding affinity and/orother biological activities of the antibody. In a preferred embodimentof the invention, a panel of antibody mutants are prepared above and arescreened for binding affinity for the antigen from the second mammalianspecies. One or more of the antibody mutants selected from this initialscreen are optionally subjected to one or more further biologicalactivity assays to confirm that the antibody mutant(s) with enhancedbinding affinity are indeed useful, e.g. for preclinical studies. Inpreferred embodiments, the antibody mutant retains the ability to bindthe antigen from the first mammalian species with a binding affinitysimilar to the species-dependent antibody. This may be achieved byavoiding altering hypervariable region residues involved in binding theantigen from the first mammalian species. In other embodiments, theantibody mutant may have a significantly altered binding affinity forthe antigen from the first mammalian species (e.g. the binding affinityfor that antigen is preferably better, but may be worse than thespecies-dependent antibody).

The antibody mutant(s) so selected may be subjected to furthermodifications, oftentimes depending on the intended use of the antibody.Such modifications may involve further alteration of the amino acidsequence, fusion to heterologous polypeptide(s) and/or covalentmodifications such as those elaborated below. With respect to amino acidsequence alterations, exemplary modifications are elaborated above. Forexample, any cysteine residue not involved in maintaining the properconformation of the antibody mutant also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant cross linking. Conversely, cysteine bond(s) may beadded to the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment). Another typeof amino acid mutant has an altered glycosylation pattern. This may beachieved by deleting one or more carbohydrate moieties found in theantibody, and/or adding one or more glycosylation sites that are notpresent in the antibody. Glycosylation of antibodies is typically eitherN-linked or O-linked N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Techniques for producing antibodies, which may be species-dependent andtherefore require modification according to the techniques elaboratedherein, follow:

A. Generation of High-Affinity Anti-VEGF Antibodies from SyntheticAntibody Phage Libraries

The invention also provides a method for generating and selecting novelhigh-affinity anti-VEGF antibodies using a unique phage displayapproach. The approach involves generation of synthetic antibody phagelibraries based on a single framework template, design of sufficientdiversities within variable domains, display of polypeptides having thediversified variable domains, selection of candidate antibodies withhigh affinity to target VEGF antigen, and isolation of the selectedantibodies.

Details of the phage display methods can be found, for example, in theprovisional U.S. Application No. 60/385,338 (filed Jun. 3, 2002) andrelating applications, the entire disclosures of which are expresslyincorporated herein by reference.

In one aspect, the antibody libraries used in the invention can begenerated by mutating the solvent accessible and/or highly diversepositions in at least one CDR of an antibody variable domain. Some orall of the CDRs can be mutated using the methods provided herein. Insome embodiments, it may be preferable to generate diverse antibodylibraries by mutating positions in CDRH1, CDRH2 and CDRH3 to form asingle library or by mutating positions in CDRL3 and CDRH3 to form asingle library or by mutating positions in CDRL3 and CDRH1, CDRH2 andCDRH3 to form a single library.

A library of antibody variable domains can be generated, for example,having mutations in the solvent accessible and/or highly diversepositions of CDRH1, CDRH2 and CDRH3. Another library can be generatedhaving mutations in CDRL1, CDRL2 and CDRL3. These libraries can also beused in conjunction with each other to generate binders of desiredaffinities. For example, after one or more rounds of selection of heavychain libraries for binding to a target antigen, a light chain librarycan be replaced into the population of heavy chain binders for furtherrounds of selection to increase the affinity of the binders.

Preferably, a library is created by substitution of original amino acidswith variant amino acids in the CDRH3 region of the variable region ofthe heavy chain sequence. The resulting library can contain a pluralityof antibody sequences, wherein the sequence diversity is primarily inthe CDRH3 region of the heavy chain sequence.

In one aspect, the library is created in the context of the humanizedantibody 4D5 sequence, or the sequence of the framework amino acids ofthe humanized antibody 4D5 sequence. Preferably, the library is createdby substitution of at least residues 95-100a of the heavy chain withamino acids encoded by the DVK codon set, wherein the DVK codon set isused to encode a set of variant amino acids for every one of thesepositions. An example of an oligonucleotide set that is useful forcreating these substitutions comprises the sequence (DVK)₇. In someembodiments, a library is created by substitution of residues 95-100awith amino acids encoded by both DVK and NNK codon sets. An example ofan oligonucleotide set that is useful for creating these substitutionscomprises the sequence (DVK)₆ (NNK). In another embodiment, a library iscreated by substitution of at least residues 95-100a with amino acidsencoded by both DVK and NNK codon sets. An example of an oligonucleotideset that is useful for creating these substitutions comprises thesequence (DVK)₅ (NNK). Another example of an oligonucleotide set that isuseful for creating these substitutions comprises the sequence (NNK)₆.Other examples of suitable oligonucleotide sequences can be determinedby one skilled in the art according to the criteria described herein.

In another embodiment, different CDRH3 designs are utilized to isolatehigh affinity binders and to isolate binders for a variety of epitopes.The range of lengths of CDRH3 generated in this library is 11 to 13amino acids, although lengths different from this can also be generated.H3 diversity can be expanded by using NNK, DVK and NVK codon sets, aswell as more limited diversity at N and/or C-terminal.

Diversity can also be generated in CDRH1 and CDRH2. The designs ofCDR-H1 and H2 diversities follow the strategy of targeting to mimicnatural antibodies repertoire as described with modification that focusthe diversity more closely matched to the natural diversity thanprevious design.

For diversity in CDRH3, multiple libraries can be constructed separatelywith different lengths of H3 and then combined to select for binders totarget antigens. The multiple libraries can be pooled and sorted usingsolid support selection and solution sorting methods as describedpreviously and herein below. Multiple sorting strategies may beemployed. For example, one variation involves sorting on target bound toa solid, followed by sorting for a tag that may be present on the fusionpolypeptide (eg. anti-gD tag) and followed by another sort on targetbound to solid. Alternatively, the libraries can be sorted first ontarget bound to a solid surface, the eluted binders are then sortedusing solution phase binding with decreasing concentrations of targetantigen. Utilizing combinations of different sorting methods providesfor minimization of selection of only highly expressed sequences andprovides for selection of a number of different high affinity clones.

High affinity binders for the target VEGF antigen can be isolated fromthe libraries. Limiting diversity in the H1/H2 region decreasesdegeneracy about 10⁴ to 10⁵ fold and allowing more H3 diversity providesfor more high affinity binders. Utilizing libraries with different typesof diversity in CDRH3 (eg. utilizing DVK or NVT) provides for isolationof binders that may bind to different epitopes of a target antigen.

Of the binders isolated from the pooled libraries as described above, ithas been discovered that affinity may be further improved by providinglimited diversity in the light chain. Light chain diversity is generatedin this embodiment as follows in CDRL1: amino acid position 28 isencoded by RDT; amino acid position 29 is encoded by RKT; amino acidposition 30 is encoded by RVW; amino acid position 31 is encoded by ANW;amino acid position 32 is encoded by THT; optionally, amino acidposition 33 is encoded by CTG; in CDRL2: amino acid position 50 isencoded by KBG; amino acid position 53 is encoded by AVC; andoptionally, amino acid position 55 is encoded by GMA; in CDRL3: aminoacid position 91 is encoded by TMT or SRT or both; amino acid position92 is encoded by DMC; amino acid position 93 is encoded by RVT; aminoacid position 94 is encoded by NHT; and amino acid position 96 isencoded by TWT or YKG or both.

In another embodiment, a library or libraries with diversity in CDRH1,CDRH2 and CDRH3 regions is generated. In this embodiment, diversity inCDRH3 is generated using a variety of lengths of H3 regions and usingprimarily codon sets XYZ and NNK or NNS. Libraries can be formed usingindividual oligonucleotides and pooled or oligonucleotides can be pooledto form a subset of libraries. The libraries of this embodiment can besorted against target bound to solid. Clones isolated from multiplesorts can be screened for specificity and affinity using ELISA assays.For specificity, the clones can be screened against the desired targetantigens as well as other nontarget antigens. Those binders to thetarget VEGF antigen can then be screened for affinity in solutionbinding competition ELISA assay or spot competition assay. High affinitybinders can be isolated from the library utilizing XYZ codon setsprepared as described above. These binders can be readily produced asantibodies or antigen binding fragments in high yield in cell culture.

In some embodiments, it may be desirable to generate libraries with agreater diversity in lengths of CDRH3 region. For example, it may bedesirable to generate libraries with CDRH3 regions ranging from about 7to 19 amino acids.

High affinity binders isolated from the libraries of these embodimentsare readily produced in bacterial and eukaryotic cell culture in highyield. The vectors can be designed to readily remove sequences such asgD tags, viral coat protein component sequence, and/or to add inconstant region sequences to provide for production of full lengthantibodies or antigen binding fragments in high yield.

A library with mutations in CDRH3 can be combined with a librarycontaining variant versions of other CDRs, for example CDRL1, CDRL2,CDRL3, CDRH1 and/or CDRH2. Thus, for example, in one embodiment, a CDRH3library is combined with a CDRL3 library created in the context of thehumanized 4D5 antibody sequence with variant amino acids at positions28, 29, 30, 31, and/or 32 using predetermined codon sets. In anotherembodiment, a library with mutations to the CDRH3 can be combined with alibrary comprising variant CDRH1 and/or CDRH2 heavy chain variabledomains. In one embodiment, the CDRH1 library is created with thehumanized antibody 4D5 sequence with variant amino acids at positions28, 30, 31, 32 and 33. A CDRH2 library may be created with the sequenceof humanized antibody 4D5 with variant amino acids at positions 50, 52,53, 54, 56 and 58 using the predetermined codon sets.

B. Vectors, Host Cells and Recombinant Methods

The anti-VEGF antibody of the invention can be produced recombinantly,using techniques and materials readily obtainable.

For recombinant production of an anti-VEGF antibody, the nucleic acidencoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated or synthesized usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to DNA encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

The antibody of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders. For yeast secretion thenative signal sequence may be substituted by, e.g., the yeast invertaseleader, a factor leader (including Saccharomyces and Kluyveromycesα-factor leaders), or acid phosphatase leader, the C. albicansglucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

The DNA for such a precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (APH) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters suitable for use with prokaryotic hosts includethe phoA promoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. No.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:1567-1575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

C. Pharmaceutical Formulations

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

D. Non-Therapeutic Uses for the Antibody

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theantigen to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the antigen to be purified, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theantigen from the antibody.

The antibodies of this invention may also be useful in diagnosticassays, e.g., for detecting expression of an antigen of interest inspecific cells, tissues, or serum.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) or a dye so that the tumor can belocalized using immunoscintiography.

In one embodiment, a method of detecting VEGF in a biological sample(e.g., tissue, blood, sera, spinal fluid) or a prepared biologicalsample can comprise the step of contacting an antibody of this inventionwith the sample and observing the anti-VEGF antibody bound to the VEGFin the sample or determining the amount of the anti-VEGF antibody boundto VEGF in the sample. In another embodiment, a method of detecting VEGFin a subject comprises the step of administering an antibody of thisinvention to the subject and observing the anti-VEGF antibody bound tothe VEGF in the subject or determining the amount of the anti-VEGFantibody bound to VEGF in the subject (e.g., human, mouse, rabbit, rat,etc).

E. Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

F. In Vivo Uses for the Antibody

It is contemplated that the antibody of the present invention may beused to treat a mammal. In one embodiment, the antibody is administeredto a nonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease to be treated with the antibody or may be used to studytoxicity of the antibody of interest. In each of these embodiments, doseescalation studies may be performed in the mammal. Where the antibody isan anti-VEGF antibody, it may be administered to a host rodent in asolid tumor model, for example.

In addition, or in the alternative, the antibody is used to treat ahuman, e.g. a patient suffering from a disease or disorder who couldbenefit from administration of the antibody. The conditions which can betreated with the antibody are many and include conditions arising fromor exacerbated by abnormal angiogenesis, e.g., by excessive,inappropriate or uncontrolled angiogenesis. For example, such conditionsinclude, cancer such as colorectal cancer and NSCLS and others describedabove and inflammatory diseases such as rheumatoid arthritis and othersdescribed above.

The following references describe lymphomas and CLL, their diagnoses,treatment and standard medical procedures for measuring treatmentefficacy. Canellos G P, Lister, T A, Sklar J L: The Lymphomas. W.B.Saunders Company, Philadelphia, 1998; van Besien K and Cabanillas, F:Clinical Manifestations, Staging and Treatment of Non-Hodgkin'sLymphoma, Chap. 70, pp 1293-1338, in: Hematology, Basic Principles andPractice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone,Philadelphia, 2000; and Rai, K and Patel, D: Chronic LymphocyticLeukemia, Chap. 72, pp 1350-1362, in: Hematology, Basic Principles andPractice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone,Philadelphia, 2000.

The parameters for assessing efficacy or success of treatment of anautoimmune or autoimmune-related disease will be known to the physicianof skill in the appropriate disease. Generally, the physician of skillwill look for reduction in the signs and symptoms of the specificdisease. The following are by way of examples.

In one embodiment, the methods and compositions of the invention areuseful to treat rheumatoid arthritis. RA is characterized byinflammation of multiple joints, cartilage loss and bone erosion thatleads to joint destruction and ultimately reduced joint function.Additionally, since RA is a systemic disease, it can have effects inother tissues such as the lungs, eyes and bone marrow.

The VEGF binding antibodies can be used as first-line therapy inpatients with early RA (i.e., methotrexate (MTX) naive), or incombination with, e.g., MTX or cyclophosphamide. Or, the antibodies canbe used in treatment as second-line therapy for patients who were DMARDand/or MTX refractory, in combination with, e.g., MTX. In one preferredembodiment, the VEGF binding antibodies of this invention areadministered to mammals who are DMARD and/or MTX refractory. Theanti-VEGF antibodies are useful to prevent and control joint damage,delay structural damage, decrease pain associated with inflammation inRA, and generally reduce the signs and symptoms in moderate to severeRA. The RA patient can be treated with the anti-VEGF antibodies of thisinvention prior to, after or together with treatment with other drugsused in treating RA (see combination therapy below). In one embodiment,patients who had previously failed disease-modifying antirheumatic drugsand/or had an inadequate response to methotrexate alone are treated withan anti-VEGF binding antibody. In another embodiment, the patients areadministered an anti-VEGF antibody of this invention pluscyclophosphamide or anti-VEGF binding antibody plus methotrexate.

One method of evaluating treatment efficacy in RA is based on AmericanCollege of Rheumatology (ACR) criteria, which measures the percentage ofimprovement in tender and swollen joints, among other things. The RApatient can be scored at for example, ACR 20 (20 percent improvement)compared with no antibody treatment (e.g., baseline before treatment) ortreatment with placebo. Other ways of evaluating the efficacy ofantibody treatment include X-ray scoring such as the Sharp X-ray scoreused to score structural damage such as bone erosion and joint spacenarrowing. Patients can also be evaluated for the prevention of orimprovement in disability based on Health Assessment Questionnaire [HAQ]score, AIMS score, SF-36 at time periods during or after treatment. TheACR 20 criteria may include 20% improvement in both tender (painful)joint count and swollen joint count plus a 20% improvement in at least 3of 5 additional measures:

1. patient's pain assessment by visual analog scale (VAS),

2. patient's global assessment of disease activity (VAS),

3. physician's global assessment of disease activity (VAS),

4. patient's self-assessed disability measured by the Health AssessmentQuestionnaire, and

5. acute phase reactants, CRP or ESR.

The ACR 50 and 70 are defined analogously. Preferably, the patient isadministered an amount of anti-VEGF binding antibody of the inventionalone or in combination with other agents for treating rheumatoidarthritis effective to achieve at least a score of ACR 20, preferably atleast ACR 30, more preferably at least ACR50, even more preferably atleast ACR70, most preferably at least ACR 75 and higher.

Psoriatic arthritis has unique and distinct radiographic features. Forpsoriatic arthritis, joint erosion and joint space narrowing can beevaluated by the Sharp score as well. The anti-VEGF binding antibodiesdisclosed herein can be used to prevent the joint damage as well asreduce disease signs and symptoms of the disorder.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, the severityand course of the disease, whether the antibody is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays. An exemplary dosingregimen for an anti-LFA-1 or anti-ICAM-1 antibody is disclosed in WO94/04188. Exemplary dosing regimens and therapeutic combinations fortreating cancer can be found in U.S. Provisional Application No.60/474,480, filed May 30, 2003.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages. Generally, alleviation ortreatment of a disease or disorder involves the lessening of one or moresymptoms or medical problems associated with the disease or disorder. Inthe case of cancer, the therapeutically effective amount of the drug canaccomplish one or a combination of the following: reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., to decrease to someextent and/or stop) cancer cell infiltration into peripheral organs;inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. In someembodiments, a composition of this invention can be used to prevent theonset or reoccurrence of the disease or disorder in a subject or mammal.

G. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the antibody. The labelon, or associated with, the container indicates that the composition isused for treating the condition of choice. The article of manufacturemay further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Incorporated by reference in their entirety are United StatesProvisional Applications from which this application claims benefit:U.S. Ser. No. 60/491,877, filed Aug. 1, 2003; U.S. Ser. No. 60/516,495,filed Nov. 1, 2003; U.S. Ser. No. 60/570,912, filed May 12, 2004; U.S.Ser. No. 60/571,239, filed May 13, 2004; U.S. Ser. No. 60/576,315, filedJun. 1, 2004; and U.S. Ser. No. 60/580,757, filed Jun. 18, 2004.

The following examples are intended merely to illustrate the practice ofthe present invention and are not provided by way of limitation. Thedisclosures of all patent and scientific literatures cited herein areexpressly incorporated in their entirety by reference.

EXAMPLES Generation of the Synthetic Antibody Phage Libraries

In general, two types of combinatorial antibody libraries have beendeveloped, distinguished by the source of repertoires. Most libraries todate are “natural” antibody libraries which use the natural repertoiresas the source for their diversity, where the genes as message RNA ofimmune cells from naïve or immunized animals or human were amplified andcloned into vector for phage display or other display technology, suchas ribosome or yeast display. The natural antibodies usually havemultiple frameworks, which together with variable CDRs sequences and therecombination of light chains and heavy chains made up the diversity ofthe library. The size of the library determines the performance of thelibrary since the repertoires are in general larger than the librarysize (Marks et al). The synthetic library, on the other hand, is a newbranch of library where the diversity is designed and built into thelibrary with synthetic DNA. Single or multiple frameworks have beenused. For single framework library, the source of the diversity solelydepends on the degeneracy of synthetic DNA designed to create thediverse CDR loops. Both the diversity design and the size of thelibraries are critical for the library performance, which can bemeasured by the affinity of the antibodies found from the libraries.

We developed a strategy of building synthetic antibody phage librariesupon the template of a single framework. Residues were selected from CDRloops that are either solvent exposed or highly variable in naturalantibody repertoires according to Kabat database and were randomized bymimicking the natural diversity using tailored codons. We also exploredrestricting the randomization to heavychains, which often contribute tothe main binding interaction with antigen among natural antibodies, andfound it was sufficient to find binders to naïve targets. One of thereasons to restrict the diversity is so that the gap between the DNAdegeneracy and practical phage library size is not overwhelmingly largeand the sequence space will be covered at sufficient density.

The first library was built on humanized 4D5 framework with a randomizedheavy chain and fixed light chain in the format of single chain Fv(ScFv). To the target antigen murine VEGF (mVEGF), many unique binderswere found.

The libraries were then generally further improved by fine-tuning thediversity of CDR-H1 and H2 or the light chain to closely mimic thenatural diversity, and by exploring the diversity design of CDR-H3 inthe Fab format of display. See FIG. 1 for illustrations of differenttypes of antibody libraries. We found libraries with different designsof CDR-H3 of similar size resulted in binders of hVEGF and mVEGF withsub-nM affinity. The affinity of these binders can be further improvedto pM range by a second step of randomizing their light chain CDRs.

For details of strategies and methods for generating synthetic antibodylibraries with a single template, see, for example, U.S. provisionalapplication U.S. Ser. No. 60/385,338 (filed Jun. 3, 2002), the entiredisclosure of which is expressly incorporated herein by reference. Thus,novel antibodies with high affinity to both human VEGF and mouse VEGFcould be found in diverse synthetic antibody phage libraries based on asingle framework template.

Example 1 G6 and B20 Derived Antibodies

(a) Selection of High Affinity Anti-VEGF Fab Clones

The selection procedures for high affinity anti-VEGF Fab clonesconsisted of various combinations of solid-supported andsolution-binding sortings. In solid-supported sortings, the antibodyphage library was panned with target antigen coated on NUNC 96-wellMaxisorp immunoplate at a concentration of 5 ug/ml. In thesolution-binding sorting method, phage library was incubated withdecreasing concentration of biotinylated antigen in solution, which thenwas captured by neutravidin coated on the 96-well Maxisorp plate (2˜5ug/ml). Decreasing concentration allowed more stringency in panning tofish for tighter binders. To the target antigen mVEGF, a two-stepsorting strategy was developed such that, in step 1, potent binders wereisolated from naïve libraries by means of the solid-supported selection,and subsequently in step 2, those stronger affinity binders could beisolated from weaker ones by the progressive solution-binding methodwith decreasing target antigen concentration. To quickly screen outthese binders, the high throughput single-spot competition binding ELISAwas used. Low amount of mVEGF (25 nM) was applied in this assay toscreen 16 clones from each library after the 3^(rd) round sorting.

As the result of combining solid-supported and solution-bindingsortings, three Fab clones, G6, B29, and C3, all from NNK library, wereidentified as high affinity binders. And after the 5^(th) round sorting,the whole library was dominated by G6 clone. Interestingly, B20, whichcame from NVT library, was found by solution-binding sorting alone. Thissuggested that more clones can be found by different strategies ofsorting methods, which may bias for different clones. With differentlibrary designs, more VEGF binding clones with distinct sequences shouldbe identified also. The four unique clones with distinct sequences werefirst characterized for their binding affinity to murine and human VEGFusing competition-binding ELISA at 25° C. IC50 data from phage bindingassays represent an estimation of their affinities, and G6 wasidentified as the highest affinity binder with IC50 at 0.5-1 nM for bothhuman and murine VEGF (FIG. 2).

(b) Activities and Properties

A series of in vitro assays were conducted to examine properties andactivities of the selected novel anti-VEGF antibodies.

Epitope Blocking Assay

First, the antibody phage clones were examined for their possiblebinding epitopes on VEGF. A phage-blocking assay was used, wherein thebindings of the phage clones (at constant concentration) to mVEGF-coatedwells were measured in the presence of either a full extracellulardomain (ECD) of KDR or a second domain of Flt-1 (Flt-1_(D2)), both atincreasing concentrations, respectively. Full ECD of KDR or Flt-1 hasseven immunoglobulin-like domains, and binds VEGF through the second andthird domain. The second domain of Flt-1 alone can bind VEGF at Kd of 2nM with known epitopes on VEGF, based on the published crystal structureof the VEGF-VEGFR complex (Wiesmann et al. (1997) Cell 91:695-704). TheKd of KDR ECD binding to VEGF is about 5 nM. We expected the binding ofthe phage clones to be reduced with the increasing addition ofreceptors, if the epitopes for an antibody on phage overlapsignificantly with the receptors.

For receptor blocking assay at protein level with purified Fab expressedfrom E. coli, (mouse or human VEGF), VEGF receptor immobilized plate bycoating baculovirus expressed Flt-1 ECD fragments (Ig domain 1-5)(Flt-1_(D1-5)) directly, or 293 cell-expressed KDR-Ig fusion, whichpresents KDR ECD (Ig domain 1-7) as Fcγ fusion captured with goatanti-human IgG Fcγ (Jackson ImmunoResearch Lab. West Grove, Pa.) coatedon the 96-well Maxisorp immunoplate and blocked with 0.5% BSA and 0.02%Tween20. Biotinylated bacterially expressed hVEGF or mVEGF at 0.2 nM wasfirst incubated with three-fold serial diluted anti-VEGF Fabs, G6,Fab-12 (the Fab of the Avastin™ antibody), or Y0317 in PBS with 0.05%Tween 20 (PBST). After 1 h incubation at room temperature, the mixtureswere transferred to a VEGF receptor immobilized plate and incubated for10 min. The VEGF-A that was not blocked by anti-VEGF was captured withVEGF receptor coated wells and detected by streptavidin-HRP conjugateand developed with TMB substrate as described above.

As shown in FIG. 3, the four clones were blocked to different extents byFlt-1_(D2) and KDR. The results of the blocking assay suggested thatthese four clones might have different binding epitopes on VEGF that arenonetheless overlapped with receptor binding epitope. The affinities ofthe antibody clones did not correlate with the efficiency of blocking byreceptor in this blocking assay. Y0959, a phage clone with known epitopewas used as a control. Among the four novel clones, G6 and B20 appearedto have epitopes that sufficiently overlapped with those for both Flt-1and KDR, since their bindings to mVEGF were significantly reduced in thepresence of the receptor fragments. The difference in the blockingefficiency is small but has been consistent with multiple assays. Oneprediction is that G6 or B20 will have epitopes that match with those ofreceptors on VEGF much better than Fab-12 or its variants, Y0317 andY0959 (Muller, Y. A., et al., (1998) Structure 6:1153-1167). Therefore,we proceeded to generate Fab protein to confirm the binding and examinethe epitope. G6 was chosen first for further study since it has thehighest affinity against both mVEGF and hVEGF.

Binding Specificity and Neutralizing Activity

To determine the binding specificity of G6 Fab clone, ELISA assays wereconducted using human, mouse, rat and rabbit VEGF-A₁₆₅ as well as VEGFhomologs including mouse and human placenta growth factor (P1GF-2),mVEGF-D and human VEGF-B. Human and murine placental growth factor(P1GF-2), murine VEGF-D, rat VEGF-A and human VEGF-B were from R&Dsystem. The tested antigens were coated on NUNC 96-well Maxisorpimmunoplate at the concentration of 2 ug/ml. Binding with increasingconcentrations of G6 Fab protein was measured by Protein G-horseradishperoxidase conjugate and substrate. For example, Fab protein wasprepared from E. coli harboring the plasmid of G6 Fab expressionconstruct under the promoter of alkaline phosphatase and secretionleader sequences stII and purified with Protein G affinity column. G6Fab binding to VEGF and its homologs were measured by direct ELISA. VEGFhomolog-coated wells (at 2 ug/ml in PBS concentration) were blocked with0.5% BSA and 0.05% Tween20 at 25° C. Fab at increasing concentrationswere incubated with the VEGF homolog-coated wells for 1 h at 25° C. andmeasured with anti-human Fab antibody horseradish peroxidase conjugatediluted in PBT buffer, then developed with TMB substrate. Solutionbinding assays were also carried out for some proteins by incubating 0.5nM of G6 Fab with increasing concentrations of a VEGF homolog for 1-2 hat 25° C., and the unbound Fab was captured with mVEGF-A-coated wellsand measured. As shown in FIG. 4, the G6 Fab protein binds equally wellto both mVEGF and hVEGF (approximately 0.6 nM and 1.4 nM, respectively).Moreover, the G6 antibody did not bind to other VEGF homologs at all,and thus is highly specific to VEGF. The G6 Fab bound rat and rabbitVEGF with similar affinity as for mVEGF (data not shown).

To test if G6 not only binds to VEGF at high affinity, but also iscapable of effectively blocking VEGF's binding to VEGF receptors,blocking assays were conducted, wherein either hVEGF or mVEGF was testedfor its binding to KDR in the presence of increasing concentrations ofthe G6 Fab clone. Also used as control were the two anti-hVEGFantibodies, Fab-12 (the Fab of Avastin™) and Y0317, that are capable ofeffectively blocking hVEGF but neither bind to mVEGF nor block itsactivities. As shown in FIG. 5, G6 effectively blocked hVEGF's bindingto KDR with an efficacy similar to that of Fab-12 or Y0317. Furthermore,G6 can also significantly block mVEGF's binding to KDR. In comparison,neither Fab-12 nor Y0317 showed any blocking effect on mVEGF.

Thus, the novel anti-VEGF antibody G6 of this invention is a highaffinity anti-VEGF antibody capable of binding and blocking VEGF fromboth human and murine species.

Cell-Based Assay

To further determine binding specificity and blocking activities of thenovel antibody G6, a cell-based assay using human umbilical veinendothelial cells (HUVECs) was conducted, wherein various anti-VEGFantibodies were tested for their abilities to block either human ormurine VEGF induced cell proliferation. Basically, 96-well tissueculture plates were seeded with 3000 Hu VECs per well and fasted in theassay medium (F12:DMEM 50:50 supplemented with 1.5% (v/v) diafilteredfetal bovine serum) for 24 hours. The concentration of VEGF used forstimulating the growth of cells was determined by first titrating toidentify the amount of VEGF that can induce 90% of maximal DNAsynthesis. Fresh assay medium with fixed amounts of VEGF (0.1 nM finalconcentration) and increasing concentrations of anti-VEGF Fab were thenadded. After 24 hours of incubation, cells were pulsed with 0.5 μCi perwell of [³H] thymidine for 24 hours, and then harvested onto 96-wellfilter plate for counting by a TopCount gamma counter. Here the DNAsynthesis was measured by incorporation of tritiated thymidine. In thisassay, the anti-VEGF antibodies serving as the control were Fab-12 andY0317.

As shown in FIG. 6, G6 antibody significantly reduced both hVEGF andmVEGF's abilities to promote HUVEC proliferation. The basic fibroblastgrowth factor (bFGF) experiment here served as a control to demonstratethat none of the anti-VEGF Fabs used in this assay had any non-specifictoxicity to the host cells.

(c) Affinity Improvement of the G6 and B20 Anti-VEGF Antibodies

Two novel antibody clones, G6 and B20, were chosen for furtherimprovement on binding affinities. To improve affinity, several selectedresidues of the light chain CDR were randomized, since both clones camefrom a library with only randomized heavy chain and a fixed light chain.Surface exposed CDR residues and residues that are highly diverse in theKabat database of natural antibody sequences were chosen. Site-directedmutagenesis was used with tailored degenerate codons to generate aminoacid diversity that mimicked the natural immune repertoire at each CDRsite on light chain (FIG. 7). Sorting was first performed by adding thelibrary on immobilized hVEGF or mVEGF on the Maxisorp 96-well plate tomaximize the recovery of binders, followed by solution sorting withdecreasing concentration of biotinylated hVEGF or mVEGF as two separatetracks of selection. Concentration of biotinylated VEGF was selectedbased on the initial affinity of the clone to gauge the pressure of thesorting. The selection pressure was also increased by incubating phagebinders with 1000 fold excess of un-biotinylated antigen in solution fordifferent lengths of time at different temperatures after initialincubation of the phage library with biotinylated VEGF and beforecapturing by neutravidin coated on 96-well Maxisorp plate.

As an example of the above-described processes, G6 having 1 nM affinitywas subject to further affinity improvement. The first round of sortingused solid-supported methods to capture all clones that still boundVEGF, and then at the second round of sorting, the phage library wasincubated with 1 nM of VEGF. Next, the solution-binding used 1 nMbiotinylated hVEGF to select most binders. Before capturing, 1 uMunbiotinylated hVEGF was added and incubated at RT for 15 min to competeoff fast off-rate binders. Then in the following rounds of sorting(3^(rd) and 4^(th)), more selection pressure was put into solutionsorting by using less biotinylated hVEGF (0.1 nM) to select and 100 nMunbiotinylated hVEGF at 37 C for 30 min or longer (2 hr or 6 hr) tocompete off high off-rate binders and fish for low off-rate binders.

The same strategy was utilized to improve the B20 clone, except thatless stringent conditions were used because of its low affinity againsthVEGF (IC50˜150 nM). The mVEGF selection track followed the sameprocedures and conditions as what were used with hVEGF. Enrichment wascalculated by the eluted phage titers ratio to selection withoutbiotinylated target. After sorting, high throughput single-spotcompetition binding ELISA as described above was used to quickly screenfor improved affinity clones.

In this assay, low amounts of hVEGF or mVEGF (10 nM) were applied toscreen 51 clones from the G6-based phage library and 110 clones from theB20-based phage library with the control of wild type G6 and B20binders. Several improved G6 clones, collectively termed G6-II variants,were selected out and showed remarkably increased binding affinity toboth human and murine VEGF by over one hundred fold by comparing thephage IC50 values with wild-type clones (FIG. 7). Clones with manyunique sequences were found that have improved affinity, which suggestedthat there are many light chains that can accommodate the heavy chain ofthe selected clones. It is also interesting to note that bindingspecificities of some clones were changed. This suggests that the lightchain is able to modulate the interaction of the heavy chain with itsantigen sufficiently to alter its specificities. As for the clonesequences, most G6-II clones were different from G6 only in their thirdlight chain CDR (CDR-L3).

To generate Fab protein or IgG protein for affinity and activity assays,we cloned the variable region into a Fab expression plasmid forexpression in an E. coli or a mammalian cell (a phagemid vector that hadbeen modified by deleting the sequence encoding the C-terminal domain ofphage coat protein p3 and adding a termination sequence for ribosomebinding about 20 nucleotides downstream from the stop codon at the endof the heavy chain's first constant domain). Fab protein was generatedby growing transformed 34B8 E. coli cells in AP5 media at 30° C. for 24h as described (Presta, L et al., (1997) Cancer Res 57:4593-4599). IgGwas purified with Protein A columns and Fab was purified with Protein Gaffinity chromatography. The production yield for Fab was typically 5-10mg/L in small scale shake flask growth and 0.5-3 g/L in fermentergrowth. IgG production was reasonably high at 10-50 mg/L small scaleculture with some clone to clone differences.

Three G6-II improved clones, G6-8, G6-23 and G6-31 (see FIG. 7 forresidue changes in light chain CDRs), were selected to make into Fabprotein for affinity measurement, epitope mapping by receptor blockingassay, and activity study in HuVEC growth inhibition assay. For affinitydeterminations of anti-mVEGF Fabs, we used surface plasmon resonanceassays on a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25° C. and 37° C. with immobilized mVEGF or hVEGF CM5 chips at˜100 response units (RU) as described (Chen, Y., et al., (1999) J. MolBiol 293:865-881). Briefly, carboxymethylated dextran biosensor chips(CM5, BIAcore Inc.) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human or murine VEGF was diluted with 10 mM sodium acetate, pH 4.8, into5 ug/ml (˜0.2 uM) before injection at a flow rate of 5 ul/minute toachieve approximately 100 response units (RU) of coupled protein.Following the injection of 1M ethanolamine to block unreacted groups.For kinetics measurements, two-fold serial dilutions of Fab (100 to 0.78nM or 3 nM to 500 nM) were injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min Association rates(k_(on)) and dissociation rates (k_(off)) were calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneously fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on).

Since the k_(on) of affinity improved G6 variants did not result insatisfactory statistics with the available binding model in BIAcoreevaluation methods, solution competition binding assays usingphage-displayed proteins were also carried out to measure the relativeaffinity of those antibodies at equilibrium. VEGF displayed on phagewere incubated with serial dilutions of Fab at room temperature for 20 hto reach equilibrium and the unbound VEGF-phage were captured with G6Fab briefly (10 min) and measured with anti-M13-HRP and substrate TMB asabove. A second format of solution competition binding assay was alsocarried out where Fab was displayed on phage and interacted withdilutions of VEGF to reach equilibrium (20 h) and unbound Fab werecaptured on VEGF coated 96-well plate and measured as above. The twoIC50s values were averaged to get binding affinities as final IC50.Binding of Fabs to mVEGF were assayed only with second format due to thelack of the reagent of mVEGF displaying phage.

From initial SPR experiments, it was noted that there was significantk_(on) improvement. For example, we found that G6-23 had the mostsignificant improvement in on-rate at 25° C. and 37° C. against bothhuman and murine VEGF, and small reduction in off-rate (FIG. 8). Thek_(on) for G6-31 improved as compared to G6 as well (data not shown).Further, the solution competition binding assays confirmed that G6-23was the most affinity-improved clone with its high affinity of IC50 of20 pM against hVEGF and mVEGF (FIG. 7).

Fluorescent quenching assays were also performed to determine the k_(on)rates of the higher affinity G6 series antibodies. Therefore, three ofthe affinity matured Fabs (G6-8, G6-23 and G6-31) were purified as Fabproteins, and their affinities for mVEGF were compared to G6 by usingfluorescence quenching in solution for the rate of association (on rate,k_(on)) (Table 1 and FIGS. 30A and B).

TABLE 1 Affinity Clone k_(on) (10⁵ M⁻¹ S⁻¹) K_(off) (10⁻⁴ S⁻¹) Kd (nM)G6 1.75 (1.93) 1.6 0.91 G6-23 56.6 1.21 0.021 G6-31 21.6 0.35 0.016 G6-816.8 1.29 0.077

The fluorescence quenching assays were performed as follows. The changeof fluorescence intensity in complexing the Fab and VEGF was used todetermine the rate of association as developed for the on-ratedetermination of Fab-12 variants and VEGF (Marvin J. S. and H. B. Loman(2003) Biochemistry 42:7077-7083). We first determined that thefluorescence intensity with an excitation wavelength of 280 nm of G6 (orvariants)-VEGF complex was lower than the sum of individual componentsusing an 8000-series SLM-Aminco spectrophometer (Thermo-Spectronic) byadding 100 nM VEGF to a stirred cuvette containing 20 nM Fab in PBS, pH7.2, at 25° C. Next, increasing concentrations of VEGF (100-400 nM) weremixed with equal volumes of 40 nM Fab in a stop-flow (Aviv Instruments)equipped spectrophometer to observe the rate of the decrease offluorescence intensity at 25° C. For each experiment, nine measurementswere performed for each concentration and fitted to a single-exponentialcurve. The observed rate was then plotted against VEGF concentrationsfor pseudo-first order analysis, and the slope is the rate ofassociation. Two to three independent experiments were carried out andthe differences were within 50%.

The estimated k_(on) of the improved clones from the SPR method were˜3-6-fold lower than the measurements by solution-based fluorescencequenching assay, whereas k_(on) measurements for parent G6 Fab from SPRwere statistically sound and within 1.2-fold difference withfluorescence quenching assays. It is possible that using BIAcore SPRtechnology for fast on-rate measurements was limited by the complex flowdynamics, or there could be some differences in protein behaviorsbetween the parent clone and affinity-improved clones, thoughaggregation problems were not observed with these proteins. Based on thefluorescence assays, the three Fabs exhibited improvements over G6 inthe on-rate by 6, 8 or 20-fold for G6-8, G6-31 or G6-23, respectively.Fab-G6-31 also exhibited an ˜4-fold improvement in the off-rate, and asa result, the affinity of Fab-G6-31 (K_(d)=16 pM) and Fab-G6-23(K_(d)=21 pM) was ˜40-60-fold improved compared to the G6 parent(K_(d)=910 pM). It was consistent with the solution phase competitionassays, which showed a 20-60-fold improvement in IC₅₀ value for G6-31(30 pM) or G6-23 (10 pM) in comparison with G6 (0.67 nM). We applied thelight chain affinity maturation strategy to five other heavy chainsequences and have observed 10- to 30-fold improvements in bindingaffinities (data not shown). Unlike G6, many improved clones adopted newsequences for all three light chain CDRs. Thus, we found that the threeG6 variants increased binding affinity to both human and murine VEGFover G6, predominantly by the increase in the rate of association (onrate or kJ. G6-23 had the highest on rate followed by G6-8 and thenG6-31. The improvement in the rate of dissociation (off rate or k_(off))was 2-3 fold for G6-23, and 8 or 13-fold for G6-31 for hVEGF or mVEGF,respectively. Compared to Fab-12 and Y0317, G6 and G6-23 hadsignificantly different binding kinetics (FIG. 32). G6-23 had similar Kdas Y0317 but a faster kinetics with fast on rate of over 10⁶ (M⁻¹s⁻¹),and moderate rate of dissociation (off rate or k_(off)) at 1−2×10⁻⁴ s⁻¹,whereas Y0317 has slow k_(on) at 3×10⁴ M⁻¹ s⁻¹, and very slow k_(off) at˜5×10⁻⁶ s⁻¹ (FIG. 32).

The solution competition binding assay used biotinylated proteinantigens equilibrated with serial dilutions of purified Fab or IgGproteins, and the unbound biotin-antigen was captured with immobilizedFab or IgG coated on Maxisorb plates and was detected with streptavidinconjugated HRP. Alternatively Fab or IgG proteins were equilibrated withserial dilutions of protein antigen, and the unbound Fab or IgG wascaptured with immobilized antigen and detected with protein A-HRP.

For the VEGF receptor blocking assay using purified Fab, VEGF receptorswere immobilized on a plate by coating Flt-1 ECD fragment (Ig domain1-5)(Flt-1_(D1-5)) directly, or by first coating with goat anti-human IgGFcγ (Jackson ImmunoResearch Lab. West Grove, Pa.) and then treating withKDR-IgG fusion receptor which presented KDR ECD (Ig domain 1-7) as adimer, on a 96-well Maxisorp immunoplate. The plate was then blockedwith 0.5% BSA and 0.02% Tween20. Sub-nanomolar concentrations ofbiotinylated hVEGF or mVEGF at 0.2 nM were incubated with three-foldserial diluted anti-VEGF Fabs, G6, G6-II (G6-8, 23, and 31), Fab-12, orY0317 in PBST. After 1 h incubation at room temperature, the mixtureswere transferred to the plate containing immobilized VEGF receptor andincubated for 10 min. The VEGF-A that was not blocked by anti-VEGF wascaptured with VEGF receptor-coated wells and detected bystreptavidin-HRP conjugate, and developed with TMB substrate asdescribed above. The results showed that, compared to the original G6,the selected G6-II Fabs indeed have increased blocking activities forboth hVEGF and mVEGF. Strong blocking had been observed with both G6 andB20 phage display clones which suggested that their binding epitopes onVEGF overlapped with the epitopes for receptors. G6 Fab was also shownto block the binding of both human and murine VEGF to Flt-1 ECD (FIG.31) efficiently. In comparison, Fab-12 can only block hVEGF but notmVEGF binding to receptor, consistent with binding specificities. AnFab-12 variant with improved affinity for hVEGF (Kd=20 pM), Y0317,inadvertently acquired some weak affinity to mVEGF (Kd ˜300 nM at 25°C., Y. Chen and H. Lowman, unpublished results) and showed a slightreceptor blocking activity to mVEGF at high concentrations.

Inhibition of VEGF activity in the endothelial cell growth assay (HuVEC)was performed as described above. Like G6, G6-II specifically inhibitedthe growth of human umbilical vein endothelial cell stimulated by humanand murine VEGF, but not the growth stimulated by human basic fibroblastgrowth factor (bFGF). The concentration required to inhibit 50% of thegrowth of the cells stimulated by both human and murine VEGF (HuVECIC50) correlated well with the affinity measured by BIAcore or solutionbinding ELISA assay at 37° C. Compared with G6, G6-23 and G6-31 showedat least 100 fold improvement in inhibiting HuVECs growth stimulated byhVEGF or mVEGF. Fab-12 and Y0317 Fabs were also measured in the sameassays to serve as controls. Fab-12 showed no measurable binding tomVEGF, whereas Y0317 Fab could bind mVEGF at 350 nM affinity. Therefore,as expected, Y0317 and Fab-12 did not show any inhibitory effects onmVEGF-mediated HUVEC growth. We showed that many G6-II variants withdifferent light chains can have improved affinity and thesolution-binding assay can also predict the outcome of the potency asinhibitor in HuVEC cell assay.

VEGF bindings of G6-23 were compared with that of G6 as well as Fab-12.As shown in FIG. 8, G6-23 has a significantly improved on-rate forbinding to both hVEGF and mVEGF. While the off-rate of G6-23 issubstantially similar to that of G6, the overall Kd of G6-23 is at leastabout 7 fold better than that of G6 for both hVEGF and mVEGF.

Example 2 Mapping of VEGF Binding Sites on G6 and G6-23 Antibodies

Functional Mapping of G6 and G6-23 by Shotgun Alanine and HomologScanning

Functional mapping of G6 and G6-23 by shotgun alanine and homologscanning were performed to identify residues that are important forbinding hVEGF and finding residues that can be improved further forbinding VEGF. We generated combinatorial phage libraries which allowedheavy chain or light chain CDR residues in separate libraries to beeither alanine or wild type (alanine scanning), or either homologousamino acid or wild type (homolog scanning).

Mutagenic Oligonucleotides for Shotgun Scanning Libraries

The following mutagenic oligonucleotides for shotgun alanine (A)- andhomolog (H)-scan library constructions to randomize CDR residues in G6and G6-23 antibodies were designed using previously described shotguncodons (Vajdos et al. (2002) J. Mol Biol 320:415-428). Equimolar DNAdegeneracies are represented in the IUB code (K=G/T, M=A/C, R=A/G,S=G/C, V=A/C/G, W=A/T, Y=C/T), and the degenerate codons are shown inbold text. All the following oligonucleotides were generated byGenentech DNA synthesis group.

Oligo Sequence H1-A GCA GCT TCT GGC TTC ACC ATT KCC GMT KMTKSG ATA CAC TGG GTG CGT CAG (SEQ ID NO: 3) H2-AAAG GGC CTG GAA TGG GTT GCA GST ATT RCTCCT GST GST GGT KMT ACT KMT TAT GCC GAT AGC GTC AAG GGC (SEQ ID NO: 4)H3-A ACT GCC GTC TAT TAT TGT GCA CGC KYT GYTKYT KYT SYT SCA KMT GCT ATG GAC TAC TGG GGT CAA (SEQ ID NO: 5) L1-AACC TGC CGT GCC AGT SMA GMT GYT KCC RCTGST GTA GCC TGG TAT CAA CAG AAA C (SEQ ID NO: 6) L2-ACCG AAG CTT CTG ATT KMT KCC GCA TCC KYT CTC KMT TCT GGA GTC CCT TCT CGC(SEQ ID NO: 7) L3-A1 GCA ACT TAT TAC TGT SMA CAA KCC KMT RCTRCT CCT SCA ACG TTC GGA CAG GGT ACC (SEQ ID NO: 8) L3-A2GCA ACT TAT TAC TGT RMA CAA GST KMT GSTRMC CCT KSG ACG TTC GGA CAG GGT ACC (SEQ ID NO: 9) H1-HGCA GCT TCT GGC TTC ACC ATT KCC GAM TWC YKG ATA CAC TGG GTG CGT CAG(SEQ ID NO: 10) H2-H AAG GGC CTG GAA TGG GTT GCA GST RTT ASCSCA KCT GST GST TWC ASC TWC TAT GCC GAT AGC GTC AAG GGC (SEQ ID NO: 11)H3-H ACT GCC GTC TAT TAT TGT GCA CGC TWC RTTTWC TWC MTC SCA TWC GCT ATG GAC TAC TGG GGT CAA (SEQ ID NO: 12) L1-HACC TGC CGT GCC AGT SAA GAM RTT KCC ASCKCT GTA GCC TGG TAT CAA CAG AAA C (SEQ ID NO: 13) L2-HCCG AAG CTT CTG ATT TWC KCC GCA TCC TWC CTC TWC TCT GGA GTC CCT TCT CGC(SEQ ID NO: 14) L3-H1 GCA ACT TAT TAC TGT VAR VAR KCC TWC ASCASC CCT SCA ACG TTC GGA CAG GGT ACC (SEQ ID NO: 15) L3-H2GCA ACT TAT TAC TGT VAR VAR GST TWC KCTRAC CCT TKG ACG TTC GGA CAG GGT ACC (SEQ ID NO: 16)Phagemid Template Vectors for Shotgun Scanning Library Constructions

(A) Phagemid pV350-2b

Phagemid pV0350-2b, which was previously used to display h4D5 Fab, ahumanized antibody against the extracellular domain (ECD) of EGF-relatedbinding receptor 2 (ErbB2), monovalently on the surface of M13bacteriophage and contained stop codon (TAA) in all three CDR in heavychain (hc), served as the template for G6 heavy chain shotgun-scanlibrary construction.

More specifically, the phagemid pV0350-2b was derived from the pS0643phagemid. The phagemid vector, pS0643 (also known as phGHam-g3, e.g.,U.S. Pat. No. 5,688,666, Example 8), contains pBR322 and f1 origins ofreplication, an ampicillin-resistant gene, an E. coli alkalinephosphatase (phoA) promoter (Bass et al., (1990) Proteins 8:309-314), asequence encoding a stII secretion signal sequence fused to residues1-191 of human growth hormone (hGH) and a sequence encoding theC-terminal residues 267-421 of protein III of M13 phage (hereinafter,cP3 or pIII). The pS0643 phagemid also contains an XbaI site and anamber stop codon following residue 191 of hGH. The stII secretion signalsequence can export a protein to the periplasm of a bacteria cell (e.g.,a light chain region (LC) of an antibody). The sequence encoding thehuman growth hormone (hGH) was removed from the pS0643 vector andreplaced with a NsiI/XbaI nucleic acid fragment encoding a humanizedanti-Her2 Fab fragment (“h4D5” sequence) ligated in frame with the stIIsecretion signal (humAb4D5-8, see Carter et al., (1992) PNAS89:4285-4289 therein or U.S. Pat. No. 5,821,337, for sequence). Theamber stop codon between the heavy chain fragment and cP3 was deleted,as this modification has been shown to increase the levels of Fabdisplayed on phage.

The h4D5 antibody is a humanized antibody that specifically recognizes acancer-associated antigen known as Her-2 (erbB2). The h4d5 sequence wasobtained by polymerase chain reaction using the humAb4D5 version 8(“humAb4D5-8”) sequence and primers engineered to give rise to a 5′ NsiIsite and a 3′ XbaI site in the PCR product (Carter et al., (1992) PNAS89:4285-4289). The PCR product was cleaved with NsiI and XbaI andligated into the pS0643 phagemid vector. The h4D5 nucleic sequenceencodes modified CDR regions from a mouse monoclonal antibody specificfor Her-2 in a mostly human consensus sequence Fab framework.Specifically, the sequence contains a kappa light chain (LC region)upstream of VH and CH1 domains (HC region). The method of making theanti-Her-2 antibody and the identity of the variable domain sequencesare provided in U.S. Pat. Nos. 5,821,337 and 6,054,297. The pS0643plasmid containing humanized 4D5 (version 8) was still further modified.For example, a herpes simplex virus type 1 glycoprotein D epitope tag(gD tag—MADPNRFRGKDLGG (SEQ ID NO:17)) was added in frame to thec-terminus of the LC using site-directed mutagenesis. Following the stopcodon downstream of the LC, a ribosome binding site and a nucleic acidmolecule encoding a BstII signal sequence were ligated to the N-terminusof the HC sequence. Consequently, the HC sequence is in frame with theC-terminal domain of the p3 (cP3), a minor coat protein of M13 phage.Thus, a Fab displayed on phage can be produced from one construct. ThisFab phagemid vector is referred to as pV0350-2b and is schematicallyillustrated in FIG. 1. The light gene in pV0350-2b was further modifiedby mutating a few other amino acid residues, e.g., Arg66 to a Gly andS93 to Ala.

To generate F(ab)′2 displayed on phage, the PV0350-4 vector was furthermodified by inserting a dimerizable leucine zipper GCN4 sequence(GRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG) (SEQ ID NO:18) between the HC andcP3 sequences by cassette mutagenesis. The GCN4 leucine zipper bringstwo sets of LC/HC-cP3 fusion polypeptides together in the E. coliperiplasm and presents the dimer on the surface of phage. This F(ab)′2phagemid vector is referred to as pV0350-4 is also schematicallyillustrated in FIG. 1.

For G6-23 heavy chain shotgun-scan library construction, phagemidpW0448-2 was constructed by introducing G6-23 CDRL3 sequence intopV0350-2b phagemid using Kunkel mutagenesis method (Kunkel et al.,(1991) Methods Enzymol 204:125-139)). Phagemid pW0448-1 was alsogenerated from pV0350-2b as template for G6 and G6-23 light chainshotgun-scanning library that contains the heavy chain variable domainof G6 and stop codon (TAA) in all three CDRs in light chain.

Construction of Shotgun Scanning Libraries

Phage-displayed libraries were constructed using Kunkel mutagenesismethod as described (Kunkel et al., 1991, supra). In total, eightlibraries were generated: hcA-G6, hcA-G6.23, lcA-G6 and lcA-G6.23 werealanine scanning libraries of heavy chain (hc) or light chain (lc) of G6and G6-23; hcH-G6, hcH-G6.23, lcH-G6, lcH-G6.23 were homolog scanninglibraries. The template containing TAA stop codon within all three heavyor light chain CDRs was simultaneously repaired during the mutagenesisreaction by the above mutagenic oligonucleotides with designeddegenerate codons. After mutagenesis, 10 ug of DNA were electroporatedinto E. coli SS320 cells (˜10¹¹ cells) (Sidhu et al., (2000) MethodsEnzymol 328:333-363), which were grown overnight at 30° C. in 2YT brothsupplemented with M13-KO7 helper phage (New England Biolabs), 50 ug/mlcarbenicillin and kanamycin. Library sizes were 2−5×10⁹. Phage wereconcentrated from the culture media by precipitation with PEG/NaCl andresuspended in phosphate-buffered saline (PBS) as described previously(Sidhu et al., 2000, supra).

Library Sorting and Screening Assays

Protein target, VEGF or anti-gD tag antibody (provided by Genentechresearch groups), was immobilized on NUNC (Roskilde, Denmark) 96-wellMaxisorp immunoplates overnight at 4° C., and before sorting, the plateswere blocked with bovine serum albumin (BSA, Sigma) for 2 hours at roomtemperature (RT). Phage libraries from the above preparation (˜10¹³phage/ml) were incubated in the target-coated immunoplates for 1 h at RTto allow for phage binding. Plates were then washed 15 times with PBSand 0.05% Tween 20 (PST) buffer, and bound phage were eluted with 0.1 MHCl for 15 minutes and neutralized with 1.0 M Tris base. Eluted phagewere propagated in E. coli XL1-blue (Stratagene) for the next round ofselection.

Individual clones selected from the second round of panning were grownin a 96-well plate overnight at 37° C. with 150 ul of 2YT brothsupplemented with 50 ug/ml carbenicillin and M13-VCS helper phage(1:2500) (Stratagene). The culture supernatants were directly used inphage competitive enzyme-linked immuno-absorbant assays (phage ELISAs)to screen functional phage-displayed G6 or G6-23 Fab variants binding totarget proteins coated on the plate (Sidhu et al., 2000, supra). Theclones exhibited both positive phage ELISA signals to VEGF antigen andanti-gD tag antibody were subjected to DNA sequence analysis.

DNA Sequencing and Analysis

The functional clones from the above screening were grown in 96-wellplates with 100 ul of 2YT broth and 50 ug/ml carbenicillin at 37° C. for2 hours. A small amount of culture supernatants (1˜2 ul) served astemplate for PCRs (GeneAmp® PCR System 9700, Applied Biosystems) toamplify the phagemid DNA fragments containing the light and heavy chaingenes with sequencing primers to add M13 (−21) universal sequences atthe 5′ end of the amplified fragments, thus facilitating the use of M13forward primers in sequencing reactions. Amplified DNA fragments in a96-well format were sequenced using Big-Dye terminator sequencingreactions and analyzed on an ABI Prism 3700 96-capillary DNA analyzer(PE Biosystems, Foster City, Calif.) by Genentech DNA sequencing group.The sequences were analyzed with the program SGCOUNT as describedpreviously (Weiss et al., (2000) PNAS USA 97:8950-8954).

The following number of functional clones in parenthesis selected fromeach library against VEGF antigen was subjected to DNA sequenceanalysis: hcA-G6 (107), hcA-G6-23 (124), lcA-G6 (111), lcA-G6-23 (102),hcH-G6 (111), hcH-G6-23 (135), lcH-G6 (106), lcH-G6-23 (97). For theclones from anti-gD tag antibody binding selection: hcA-G6 (108),hcA-G6-23 (130), lcA-G6 (116), lcA-G6-23 (98), hcH-G6 (120), hcH-G6-23(122), lcH-G6 (111), lcH-G6-23 (102).

Fab G6 and G6-23 Point Mutants and Affinity Measurements

To generate G6 and G6-23 Fab mutants for affinity measurements, we useda previously modified Fab expression plasmid from phage-displayed vectorwith an E. coli alkaline phosphatase (phoA) promoter (Presta et al.,(1997) Cancer Res 57:4593-4599). Each point mutant was constructed usingthe Kunkel site-directed mutagenesis method (Kunkel et al., 1991, supra)with oligonucleotides designed to have point mutation within CDRs. Formutant productions, expression plasmids were transformed into 34B8 E.coli cells, and a single colony was picked and grown in completeC.R.A.P. medium (Presta et al., 1997, supra) supplemented with 25 ug/mlcarbenicillin at 30° C. for 24 h. The expressed recombinant proteinswere purified through a Protein G high trap column (Amersham Pharmacia)and quantitated by UV absorption at 280 nm (Presta et al., 1997, supra).The binding affinities of G6 and G6-23 Fab mutants were evaluated usingcompetitive solution binding ELISA with hVEGF displaying phage asdescribed. The fold reduction in wild-type VEGF phage binding activitydue to each point mutation was determined by dividing the IC₅₀ for theG6 or G6-23 point mutant by the IC₅₀ for wild-type G6 or G6-23 Fabrespectively.

BIACORE™ Binding Analysis for FabG6 and G6-23 Variants

For binding kinetics, surface plasmon resonance (SRP) measurement with aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) was used as previouslydescribed (Karlsson & Fält, (1997) J. Immunol Methods 200:121-133).Carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) wereactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Human VEGF (hVEGF) was diluted with 10 mMsodium acetate, pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at aflow rate of 5 ul/minute to achieve approximately 100 response units(RU) of coupled protein followed by the injection of 1M ethanolamine toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of G6 or G6-23 Fab variants (0.7 nM to 100 nM) were injectedin PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate of 25 ul/minAssociation rates (k_(on)) and dissociation rates (k_(off)) werecalculated using a simple one-to-one Langmuir binding model (BIAcore™Evaluation Software version 3.2). The equilibrium dissociation constant(Kd) was calculated as the ratio k_(off)/k_(on). Based on the selectionof the phage library, two residues in heavy chain CDR2 appeared toprefer to be alanine or homologous amino acid: HC-Y58 preferred to bealanine, HC-I51 preferred to be valine for binding VEGF. We thereforegenerated mutant protein Fab with a single mutation to confirm thefinding from phage libraries. The top graph in FIG. 9 shows that theBIAcore analysis of mutation G6-23-Y58A (G6-23.1) (50 nM) binding tohuman VEGF coated chip improved the binding on-rate compared to G6-23,which already had a much improved on-rate compared to G6. The lowergraph showed the BIAcore analysis of mutant G6-Y58A (G6-1) or G6-I51V(G6-2) to VEGF coated chip. Both had improved on-rate compared to G6. Infact, the double mutant (Y58A/I51V) had an additive effect on theon-rate improvement for G6 and G6-23.

Results of Shotgun Scanning of G6 and G6-23

The results of both shotgun scanning on G6 and G6-23 antibodiesindicated that heavy chain CDR side-chains dominated the bindinginteractions with hVEGF. When mapped onto the X-ray crystal structure ofG6 Fab, the functionally important residues in combination represent aportion of the structural epitope, which is defined by the residues indirect contact with hVEGF. The shotgun alanine-scan results revealed thefunctional epitope comprising the solvent-exposed ridge, which wascomposed of residues Y32, W33, G54, V96, F97, F98, L99, and Y100a on theheavy chain and Y49 on the light chain. Interestingly, the numbers offunctionally important residues revealed by the shotgun homolog-scanwere significantly smaller and contained within the group of residuesthat were important functionally in the shotgun alanine-scan. These hotspots are W33, G54, V96, F97, and L99, which constitute a small patchthat overlapped in both scans, suggesting that this surface makesprecise contact with the antigen hVEGF that even homologous amino acidsubstitutions were disruptive. G55 was substituted with alanine only inthe homolog scan and it was a highly disruptive, so we believe that G55is also within this patch.

Other functional important residues revealed by both scans on the heavychain, G50 and T52, are not part of the solvent-exposed surface of G6since they are buried inside according to the structural information,acting as scaffolding side-chains that pack against residues in thefunctional epitope in order to hold this epitope in a binding-competentconformation (C. Wiesmann, unpublished results). As for F95 in CDR-H3 ofG6, definitely, alanine substitution at this position in alanine-scanwas disruptive; nonetheless, this residue is capable of toleratingtyrosine substitution in homolog-scan, suggesting that its role is inmaintaining the structure integrity of the antigen-binding site, whichhas also been confirmed with structural information.

For Y32, F98, and Y100a, the structural information demonstrated thatthese surface residues were critical and represented 30% of total heavychain buried surface areas upon binding hVEGF. The alanine substitutionsat these residues were definitely disruptive for affinity withsignificant function ratios (F_(wt/mut) value) greater than 30, yetinterestingly, they tolerated homologous amino acid substitutions (Tyrfor Phe and vice versa), suggesting that the aromatic rings for thesepositions were making the important interactions with hVEGF antigen, andadding or removing the hydroxyl group was not making much impact, whichwas distinct from the above scaffolding residues.

Both shotgun scans identified new mutations in the heavy chain forfurther affinity improvement, which were I51V and Y58A. These intriguingobservations were verified through producing Fab point mutants, andtesting binding activity with competitive ELISA and BIAcore™ (FIG. 23and FIG. 18). Because I51 and Y58 are not the members of structuralepitope (C. Wiesmann, unpublished results), it was believed that theaffinity improvement, especially in on-rate, with single or doublemutation was not from the introduction of new contact with antigenhVEGF, but probably through optimizing the structure integrity of CDR-H2loop, to make it more competent for antigen binding.

Based on the structural epitope, G6 light chain CDR residues represented25% of total structural epitope surface (878 Å²) (C. Wiesmann,unpublished results). Interestingly, the only residue that showedfunctional importance in both shotgun scanning was Y49 in CDR-L2, whichonly stood for 2% of the total structural epitope surface, suggestingthat most structural epitope residues in the light chain were notinvolved in the energetic contact with hVEGF antigen. This readoutimplies that G6 light chain is more important structurally thanfunctionally since it still retained the same sequence as 4D5, thetemplate used for displaying antibody library on the phage where G6 wasisolated from in the first place (Carter et al., 1992).

Example 3 G6 and G6-23 Derived Antibodies

(a) Libraries for Selection

Additional anti-VEGF antibodies were obtained by sorting phage from theshotgun alanine and homolog scanning libraries described in Example 2.Specifically, at particular residues, scanned residues were allowed tovary as either wild type or alanine (alanine scan), or either wild typeor a homologue residue (homolog scan). (FIGS. 14A and B). For the G6,shotgun alanine and homolog scanning were performed on the G6 lightchain and heavy chain separately, resulting in four libraries. For G623,shotgun alanine and homolog scanning were performed on the G623 lightchain, and shotgun homolog scanning was performed on G623 heavy chain,resulting in three libraries. A G623 shotgun alanine scanning librarywas not prepared because, as discussed above, 6623 is a high affinityantibody, with most of its residues critical for binding located in theheavy chain. Mutating heavy chain residues to alanine, which meanstruncating the heavy chain amino acid side chains, would most likelydisrupt binding and result in lower affinity antibody variants.

(b) Selecting Anti-VEGF Antibodies

In the first round, a plate sorting strategy was used for selection.Human VEGF (hVEGF, provided by Genentech Research Groups) wasimmobilized on 96-well Maxisorp immunoplates (NUNC) by incubating wellswith 5 ug/ml of protein target over night at 4° C. The plates wereblocked with 1% Bovine Serum Albumin (BSA, Sigma) for 30 min in RT,after which 1% Tween20 was added for additional 30 min. The phagelibraries described above were incubated on the hVEGF coatedimmunoplates at a concentration of ˜1E13 phage/ml for 1 h in RT underagitation, to allow binding of the phage displayed Fab to the target.After binding, plates were washed 15 times with PBS supplemented with0.05% Tween20. Bound phage were eluted by incubating wells with 0.1M HClfor 30 min. Elutions were neutralized with 1.0 M Tris base, pH 11. E.Coli XL1-blue (Stratagene) were infected with the eluted phage andM13-K07 helper phage (New England Biolabs). The phage was propagatedover night for the next round of selection.

The bacterial culture was centrifuged at 8000 rpm for 10 min in aSorvall GSA rotor at 4° C. and the supernatant collected. The phage wasprecipitated from the media by addition of 1/5 volume of PEG/NaCl. After5 min incubation on ice the phage containing culture media wascentrifuged at 10 krpm for 15 min in a Sorvall GSA rotor at 4° C. andthe supernatant discarded. The remaining phage pellet was resuspended inPBS and centrifuged at 15 krpm for 5 min in a SS-34 rotor at 4° C. topellet insoluble matter. The supernatant was transferred to a new tubeand the phage concentration was estimated by measuring the absorbance at268 nm.

The following rounds of sorting were performed by a solution-phasesorting method with increasing stringency. Purified phage was incubatedwith biotinylated hVEGF for 1 hour at RT in PBS with 0.05% Tween20 and0.5% Superblock (Pierce). After three to ten-fold dilution withsuperblock, the mixture was added to neutravidin coated wells blockedwith 1% BSA (Sigma) and Tween20. The hVEGF-conjugated biotin was allowedto bind the immobilized neutravidin for 10 min. After capture the platewas washed 10-15 times with 0.05% Tween20 in PBS. To study thebackground, i.e. nonspecific binding on the phage to neutravidin, acontrol reaction lacking the biotinylated hVEGF was added to a coatedwell. The stringency was increased each round by decreasing antigenconcentration in solution, as well as phage concentration or increasingtemperature. Increasing the number of washes is also a way to achievemore stringent conditions. For the third and fourth round of sortingnon-biotinylated hVEGF was added to the reaction mix of biotin-hVEGF andlibrary phage to compete off low affinity binders. By adding 1000 foldexcess of competitor for 30 min in 37° C. before capturing, stringencycould be increased additionally in the last rounds in order to selectfor high affinity binders. Manipulating binding and capture times couldfurther increase the stringency from round to round.

For G623 the starting concentration of biotinylated hVEGF was 1 nM forthe heavy chain shotgun library and 0.2 nM for the light chainshotgun-scanning library. The concentration was decreased to 0.5 nM and0.1 nM respectively in the last round. For G6, with lower startingaffinity than G623, library sorting was performed with less stringentstarting conditions; 5 nM for heavy chain libraries and 2 nM for thelight chain libraries, which was decreased to 1.75 and 0.5 nMrespectively in the last round of sorting.

To study the enrichment of hVEGF specific Fabs displayed on the phage,90 ul E. Coli XL-Blue (Stratagene) was infected with 10 ul phage elutionfrom the captured library binding reactions as well as the backgroundmixtures for 30 min at 37° C. A 5 ul culture was plated out oncarbencillin supplemented plates and incubated at 37° C. over night. Thenumber of phage particles in the elution could be calculated from thenumber of colonies from each elution. After the last two rounds ofsorting, 50 ul of the above mentioned culture of phagemid-containingbacteria, was plated out on carbencillin-supplemented plates and grownover night at 37° C. The resulting clones were subject to screening.

(c) Single Spot Competitive ELISA

To screen for high affinity clones a high-throughput single pointcompetitive ELISA assay in 96-well format was performed. For bothantibodies approximately a hundred clones from the last two rounds ofselection were picked randomly and screened in this assay. Phagemidcontaining E. Coli XL1-blue clones were grown in 400 ul rich 2YT broth,supplemented with carbencillin and M13-KO7 helper phage (New EnglandBiolabs) by shaking over night at 37° C. The culture supernatant of eachclone was diluted five times with 0.05% Tween20 and 0.5% BSA in PBS withaddition of hVEGF as well as without. After 1 h incubation by shaking inRT, 80 ul of the reactions were transferred to hVEGF-coated immunoplatesand incubated for 10 min. The plate was washed 8 times with 0.05%Tween20 in PBS and incubated 30 min with 100 ul anti-M13 antibodyhorseradish conjugate (New England Biolabs) diluted 5000 times in PBSsupplemented with 0.05% Tween20 and 0.5% Bovine Serum Albumin (Sigma).The plates were washed an additional 8 times and developed with TMBsubstrate for 5 min. The reactions were stopped with 1.0 M H₃PO₄ andread spectrophotometrically at 450 nm. To estimate the relativeaffinity, the ratio of the optical density in the presence of hVEGF tothat of the absence of hVEGF was calculated. A low ratio suggests thatmost Fab displayed on on the phage bound hVEGF in solution. As aconsequence little Fab can bind to the immobilized hVEGF on theimmunoplate. Clones showing lower ratio than wild type (WT) G6 or G623were selected and, their supernatants used to infect XL1-blue. Phagemidcontaining bacteria were streaked out on carbencillin plates and grownover night at 37° C. for sequencing.

The selection of the G6 heavy chain libraries resulted in no more thanthree unique sequences out of the 33 clones analyzed, suggesting thatmost G6 heavy chain changes result in loss of binding affinity.Furthermore, all unique heavy chain variants derived were from thehomolog scan library. It seems that alanine mutation of G6 heavy chainresidues can result in a dramatic loss of binding. This intolerance tochanges in amino acid sequence selection supports the view that G6 heavychain harbors many key residues for VEGF binding. The single spot assayresult is summarized in Table 2.

TABLE 2 Single Spot Summary G623 66 Library LC Ala LC Hom HC Hom LC HCAla and Hom Ala and Hom Number of 84 84 88 88 96 Clones ScreenedSelection Clones with significantly lower ratio Criteria (Signal +hVEGF/Signal-h VEGF) than WT Cut Off Cut off = 2 fold reduction Cut off= 5 Cut off = WT fold ratio reduction Number of 33 17 29 56 33 Cloneswith ratio <WT Number of 17 12 18 53  3 unique clonesResults from Single spot assay of G6 and G623 variants. Similarselection criteria was used for both antibody libraries. However, lowercutoffs were used for the G6 LC library compared to the G6 HC library asHC variants were expected to show less affinity improvement compared toWT than LC variants. G6 alanine and homolog libraries were pooled beforescreening the heavy chain and light chain variants. The number of uniqueclones from the selected pool was determined by DNA sequencing. LC=LightChain. HC=Heavy Chain. WT=Wild Type. Hom=Homolog scanning library.Ala=Alanine scanning library

(d) DNA Sequencing and Analysis

Clones selected from the single spot screening were grown in 96-wellplates with 100 ul 2YT broth supplemented with carbencillin for 2 hours.1 ul of the culture was subjected to PCR (GeneAmp® PCR System 9700,Applied Biosystems) in order to amplify the DNA encoding the light chainor heavy chain of the different clones. The primers used in the PCRreaction were designed to add M13 universal sequences at the 5′ end ofthe amplified fragment. By adding this sequence, M13 forward primerscould be used in the following sequencing reactions. After amplificationin 96-well format, the DNA fragments were sequenced by Genentech DNAsequencing group using Big-Dye terminator sequencing reactions andanalyzed with an ABI Prism 3700 96-capillary DNA analyzer. (PEBiosystems, Fosters City, Calif.). The heavy and light chain sequenceswere aligned and identical sequences eliminated. Based on theirsequence, clones were selected for affinity determination by IC50measurement.

(e) Affinity Measurement of Phage Clones

The IC50 values for the phage clones were determined by competitivephage ELISA. A single colony of the selected clones was grown in 5 ml2YT media supplemented with 50 ug/ml carbencillin for 5 hours. Theculture was then infected with M13-K07 helper phage (New EnglandBiolabs) for 1 h and transferred to a 25 ml culture supplemented with 50ug/ml carbencillin and 50 ug/ml kanamycin. The phage was propagated overnight at 37° C. After phage purification as described above, the phagewas serially diluted with 0.05% Tween20 and 0.5% BSA in PBS andincubated on a hVEGF-coated immunoplate to assess antigen binding atdifferent phage concentrations. The phage dilution that gave ˜70%saturating signal was used in the IC50 determination assay. Phage wasincubated with increasing concentration of hVEGF over night at 37° C.The unbound phage was captured on an hVEGF coated immunoplate for 10min. The plate was washed, and the bound phage was detected with ananti-M13 antibody horseradish conjugate (New England Biolabs) followedby TMB development, as previously described. The hVEGF concentrationthat inhibited 50% of phage binding to the immobilized antigenrepresented the IC50. The signals, which indicate bound phage, wereplotted against the hVEGF concentration, and the data was fitted to acompetitive binding curve by non-linear regression, enabling IC₅₀determination.

FIGS. 34 and 35 show the IC₅₀ values of G6 and G6-23 variants,respectively, as well as the alterations in sequences compared to G6 orG6-23. From the G6 pool, many binders with apparent better affinity thanwild type G6 were identified. Many more affinity-improved G6 variantswere derived from the light chain homolog library (FIG. 34A). However,two higher affinity G6 variants were selected from the heavy chainhomolog scan library (FIG. 34B).

Some G6 variants showed up to a hundred-fold affinity improvement,according to the IC₅₀ determinations. Of the few G6 heavy chain variantsselected, they differed in only a few residues from wild type G6. Theseheavy chain mutations were located in CDR-H1 and CDR-H2. The conservednature of the heavy chain, and CDR-H3 in particular, confirmed that mostresidues important for G6 binding to hVEGF are present in this region.Mutations were more abundant in the G6 light chain variants. They weremainly located in CDR-L1 and CDR-L3 regions, indicating that changingCDR-L2 residues impairs binding instead of improving affinity. Acombination of mutations in CDR-L1 and CDR-L3 seem to be favorable forbinding, allowing significant affinity improvement compared to wildtype.

Most G623 variants showed similar or slightly reduced binding affinitycompared to wild type according to their IC₅₀ values (FIG. 35). Thehighest affinity variants were derived from both homolog and alaninelight chain shotgun library. Only a small number of high affinitybinders were identified in the heavy chain shotgun scan pool. As for G6,heavy chain residues were conserved, and only few mutations presentamong the clones studied. The light chain variants also showed a similarmutation-pattern as G6 variants, with most changes located in CDR-L1 andCDR-L3.

Example 4 Alanine Scanning of hVEGF to Map Anti-VEGF Antibody BindingSites

To understand the molecular basis of the cross reactivity of G6 andG6-23 and to map their functional epitopes, the relative bindingaffinities of G6 or G6-23 Fab for individual alanine-substituted hVEGFmutants versus wild type hVEGF were measured using hVEGF displayingphage ELISA as previously described (Muller et al., (1997) PNAS USA94:7192-7197). hVEGF was mutated at sites near the binding epitopes ofFlt-1, KDR, the Avastin™ antibody and Y0317 with Kunkel mutagenesismethod and phage displaying the individual mutant was generated asdescribed previously (Muller et al., (1997) PNAS USA 94:7192-7197).Solution binding phage ELISAs were used to determine the relativebinding affinity of each mutant VEGF mutant versus wild type (wt) VEGFon Fab G6-23, G6-23. Briefly, 96-well Maxisorp immunoplates (NUNC) werecoated overnight at 4° C. with G6 or G6-23 Fab at a concentration of 2ug/ml in PBS, and blocked with PBS, 0.5% BSA, and 0.05% Tween20 (PBT)for 2 h at room temperature. Serial dilutions of phage displaying hVEGFmutants in PBT were first incubated on the G6-Fab-coated plates for 15min at room temperature, and the plates were washed with PBS, 0.05%Tween20 (PBST). Bound phage were detected with anti-M13 monoclonalantibody horseradish peroxidase (Amersham Pharmacia) conjugate diluted1:5000 in PBT, developed with 3,3′,5,5′-tetramethylbenzidine (TMB,Kirkegaard & Perry Labs, Gaithersburg, Md.) substrate for approximately5 min, quenched with 1.0 M H₃PO₄, and read spectrophotometrically at 450nm. The relative IC₅₀ values (IC_(50,Ala)/IC_(50,wt)) to represent thefold of reduction in binding affinity, and evaluate the energeticcontribution of individual side-chains of hVEGF for interacting with G6or G6-23 Fab were calculated.

The ratios of IC50 represented the energetic contribution of theindividual side chain in the interaction with G6 Fab (FIG. 19). Thegeneral folding of alanine mutants, especially ones that had significantreduction in binding G6, was verified by its near-wild type binding toother molecules that have distinct epitopes on VEGF, such as theAvastin™ antibody, Y0317 or Flt-1, as mapped previously (FIG. 19). It isevident that G6 had a distinct epitope as compared to hybridoma derivedAvastin™ antibody. All functionally important hVEGF residues for bindingG6 were conserved between human and murine VEGF, thus explaining in partits cross reactivity. The Fab of the Avastin™ antibody (Fab-12) andY0317, on the other hand, lost most of their binding to VEGF uponalanine substitution at residue Gly88, which is Ser in mVEGF. Tovisualize the epitope on a VEGF molecule, functionally importantresidues for binding G6 were highlighted on the surface of hVEGF crystalstructure according to their relative affinities for G6. G6 epitopemapped to a patch that was conserved between human and mouse VEGF anddotted with a few energetically important residues, Phe17, Tyr21, Tyr,Ile83 and Gln89, which clustered in close proximity. This is indicativeof a functional epitope with an interaction “hot spot”.

We next compared the functional epitope of G6 to the structural epitopeof Fab-12 (same as Y0317) or Flt-1D2, the VEGF residues that becameburied upon forming a complex with these molecules based on the crystalstructure of the complex. Interestingly, the functional epitope of G6 onVEGF matches the structural epitope for Flt-1D2 more closely than doesthe structural epitope of Fab-12. Further, G6 shared a similar hot spotas Flt-1 for VEGF since residues Phe17, Tyr21 and Tyr25 were highlyimportant energetically for both interactions (FIG. 19). Fab-12 orY0317, on the other hand, centered on the non-conserved and functionallyimportant residue, Gly88, which is believed to be the reason for itslack of binding to mVEGF. It appeared that phage library derivedantibody G6 targeted a conserved epitope on VEGF in a nearly identicalfashion as the VEGF receptor, while a hybridoma for hVEGF avoidedgenerating a self-reactive anti-mouse antibody. There was, however,sufficient overlap between the two epitopes of G6 and Fab-12 as thebinding of G6 and Fab-12 (Y0317) to hVEGF were mutually exclusive (datanot shown).

Comparing the important sites on hVEGF for binding G6 and G6-23indicated that the residues contributing most energetically, F17, Y21,and Y25 on 20's helix, and Q89A on 80's loop, remained the same, exceptthe impact of residues on binding seemed to change (FIG. 19). Forexample, residues Y21 and Q89 when changed to alanine became a biggerhit for G6-23 than for G6. There were sites that modestly decreased itsrelative impact of its side chain in binding to G6-23 as compared to G6,e.g., 183, H86, and I91A.

There were no new functionally important residues observed for theaffinity-improved version, G6-23, yet a shuffling of energeticcontributions among these functionally important residues in bothantibodies, which was consistent with the finding structurally thatidentical positions of hVEGF were buried upon complexing with G6 orG6-23 (structural data below). When mapping these functionally importantresidues along with other moderate contributors on hVEGF structure, theyappeared as a contiguous patch within the structural epitope of Flt-1D2,which were highly conserved between human and mouse VEGF, which explainsthe fact that both antibodies had equal affinity for both VEGF. Bycomparison of the footprint of both structural and functional epitopesfor G6 on hVEGF structure, it clearly showed that heavy chain CDRresidues were the ones making contact with the positions on VEGF wherealanine mutations were disruptive, such as the 20's helix and 80′ loopof hVEGF, whereas light chain CDR residues contact the 60's loop ofhVEGF where the side chains were not functionally as important.

The dilutions of phage that produced the sub-maximal binding signal(50-70%) were used in the solution competition assay where wild type ormutant hVEGF phage in PBT buffer were first incubated with increasingconcentration of competing G6 or G6-23 Fab for 1-2 h at roomtemperature, then the mixtures were transferred to G6-Fab-coated platesto capture the unbound phage for 15 min, and bound phage were detectedas described above. Competition curves were fitted with a four-parameternon-linear regression curve-fitting program (Kaleidagraph, SynergySoftware) to determine the IC₅₀ values which were calculated as theconcentration of G6 or G6-23 Fab in solution binding stage thatinhibited 50% of the phage from binding to immobilized G6 Fab. The ratioof the IC₅₀ of mutant versus wild type hVEGF is the relative folddifference in binding affinities. For mutants that have severe reducedbinding to G6-Fab-coated plate, Fab12 (Presta, 1997) or Flt-1_(D1-5)were used as coat to capture to mutant phage after incubations with G6or G6-23 Fab. Binding of hVEGF-phage to G6, G6-23 Fab, Fab12, Fab andFlt-1_(D1-5) were mutually exclusive as tested with wild type VEGFphage.

Example 5 Structural Mapping of VEGF Binding Sites on G6, G6-23 and B20and G6 and B20 binding sites on VEGF by Crystallography

Expression, Purification Crystallization and Structural Analysis

Residues 8-109 of human VEGF were expressed, refolded, and purified aspreviously described (Christinger, H. W., et al., (1996). Prot. Struct.Funct. Genet. 26, 353-357).

G6 Fab was expressed in E. coli and the cell paste was thawed into PBS,25 mM EDTA, 1 mM PMSF. The mixture was homogenized and then passed twicethrough a microfluidizer. The suspension was then centrifuged at 12 krpm for 60 min. The protein was loaded onto a Protein G columnpreviously equilibrated with PBS at 5 ml/min. The column was washed withPBS to baseline and then eluted with 0.58% acetic acid. Fractionscontaining G6 Fab were pooled and loaded directly onto a SP-sepharosecolumn equilibrated with 20 mM MES, pH 5.5. The protein was eluted witha salt gradient of 0 to 0.25 M NaCl.

G6 eluted from the SP-sepharose column was mixed with hVEGF8-109 andfurther purified over a Superdex 200 column equilibrated with 30 mMTris.Cl pH 7.5 and 0.4 M NaCl. Fractions containing the complex werepooled, concentrated and used in crystallization trials. Crystals weregrown at 19° C. using vapor diffusion method in sitting drops.Crystallization buffer containing 2.0 M ammonium sulfate and 5%iso-propanol was mixed in equal volume with protein solution (8 mg/mlprotein). Crystals appeared after 3 days and belonged to space groupP3121 with cell dimensions of a=117.9 Å and c=212.6 Å. These crystalforms contained 1 complex comprising of a VEGF dimer and 2 Fab moleculesin the asymmetric unit.

The crystals were soaked in mother liquor, dipped in artificial motherliquor containing 25% glycerol and flash frozen in liquid nitrogen. A2.8 Å data set was collected on an SSRL Synchrotron Source on beam line9-1. The data were reduced using programs DENZO and SCALEPACK(Otwinowski, Z. (1993). DENZO. In Data Collection and Processing, L.Sawyer, N. Isaacs, and S. Bailey, eds. (Warrington, UK: 1993)).

Structure Determination and Refinement

The structure was solved by molecular replacement using the coordinatesof VEGF (from PDB code 1FLT), constant and variable domains of theantibody in 1BJ1 (Brookhaven database) and program AMoRe (CCP4 1994).Model building was done with program 0 and refinement with Refmac (CCP41994). The final Rvalue and Rfree are 19.87% and 23.92%, respectively.

The following programs were used to calculate the surface areas ofinteraction RESAREA version 3.2: 05/08/93 and AREAIMOL version 3.2:19/12/95. These programs are part of the CCP4 suite CollaborativeComputational Project, Number 4. 1994 (“The CCP4 Suite: Programs forProtein Crystallography” Acta Cryst. D50, 760-763).

The surface area of each residue of an anti-VEGF antibody that is buriedin VEGF (Å²) is reported below together with the percentage of the totalsurface area of the residue that is buried. Also reported is the surfacearea of each residue of VEGF antibody that is buried in VEGF (Å²) isreported below together with the percentage of the total surface area ofthe residue that is buried. See values for G6:VEGF, G6-23:VEGF,Fab-12:VEGF, YADS-1:VEGF and YADS-2:VEGF complex below. In all cases,because VEGF is a dimer, the residue numbers of VEGF referring tomonomer 1 (of the VEGF dimer) are 8-109 and residue numbers of VEGFreferring to monomer 2 (of the VEGF dimer) are 1008-1109. The firstcolumn in each table below recites the residue numbers of the proteinbeing examined (either VEGF or an anti-VEGF antibody) (e.g., for section(a) below, PHE A 17 refers to F17, LYS A1048 refers to K48; MET A1081refers to MET 81 of VEGF). The second column recites the buried surfaceof that residue (Å²). The third column recites the buried surface forthat residue as a percentage of the surface area of the whole residue.

(a) VEGF:G6 Complex

TABLE 3 Residues of VEGF in contact with G6 buried Residue Nr surfaceburied surface in % PHE A 17 17.00 54.84% of 31.00 MET A 18 59.00 57.84% of 102.00 TYR A 21 62.00 100.00% of 62.00  GLN A 22 65.00 48.51% of 134.00 TYR A 25 54.00 70.13% of 77.00 ASP A 63 54.00 68.35%of 79.00 GLU A 64 31.00  23.66% of 131.00 LEU A 66 33.00 73.33% of 45.00CYS A 104 10.00 34.48% of 29.00 PRO A 106 49.00  48.51% of 101.00 LYSA1048 40.00 86.96% of 46.00 MET A1081 17.00 94.44% of 18.00 ILE A108325.00 89.29% of 28.00 LYS A1084 1.00  0.88% of 114.00 PRO A1085 2.00 3.77% of 53.00 HIS A1086 103.00  53.65% of 192.00 GLN A1087 19.00 12.18% of 156.00 GLY A1088 13.00 38.24% of 34.00 GLN A1089 119.00 88.15% of 135.00 HIS A1090 22.00  20.37% of 108.00 ILE A1091 30.0047.62% of 63.00 CHAIN A DIFF-AREA: 825.0 (7.47% of 11043.0 total AREAfor this chain)

TABLE 4 Residues of G6 in contact with VEGF Residues 1:211 refer to thelight chain. Residues 1001:1223 refer to heavy chain Residue Nr buriedsurface buried surface in % ASP B 28 38.00  38.00% of 100.00 SER B 3023.00 47.92% of 48.00 TYR B 49 19.00 65.52% of 29.00 PHE B 53 25.00 21.55% of 116.00 TYR B 92 70.00 94.59% of 74.00 THR B 93 19.00 38.00%of 50.00 SER B1030 17.00 21.52% of 79.00 ASP B1031 96.00  84.21% of114.00 TYR B1032 24.00 41.38% of 58.00 TRP B1033 52.00 77.61% of 67.00ILE B1051 14.00 29.79% of 47.00 PRO B1053 38.00 97.44% of 39.00 ALAB1054 26.00 42.62% of 61.00 GLY B1055 49.00 96.08% of 51.00 GLY B105639.00 97.50% of 40.00 TYR B1057 38.00  22.35% of 170.00 PHE B1099 2.00100.00% of 2.00  PHE B1101 101.00  90.99% of 111.00 PHE B1102 109.00 84.50% of 129.00 LEU B1103 10.00 62.50% of 16.00 PRO B1104 5.00 13.16%of 38.00 TYR B1105 42.00 66.67% of 63.00 CHAIN B DIFF-AREA: 856.0 (4.44%of 19280.0 total AREA for this chain) TOTAL DIFF-AREA: 1681.0 (5.54% of30323.0 total AREA over all chains)

(b) VEGF:G6-23 Complex

TABLE 5 Residues of VEGF in contact with G6-23 Residues 8:109 relate tomonomer 1 (of the VEGF dimer). Residues 1008:1109 to monomer 2 (of theVEGF dimer) Residue Nr buried surface buried surface in % LYS A 48 35.0064.81% of 54.00 MET A 81 23.00 95.83% of 24.00 ILE A 83 18.00 90.00% of20.00 LYS A 84 1.00  1.04% of 96.00 PRO A 85 2.00  4.76% of 42.00 HIS A86 114.00  55.34% of 206.00 GLN A 87 18.00  11.18% of 161.00 GLY A 8814.00 42.42% of 33.00 GLN A 89 116.00  87.88% of 132.00 HIS A 90 22.00 16.30% of 135.00 ILE A 91 33.00 49.25% of 67.00 PHE A1017 24.00 68.57%of 35.00 MET A1018 56.00  46.67% of 120.00 TYR A1021 72.00 100.00% of72.00  GLN A1022 68.00  56.20% of 121.00 TYR A1025 58.00 67.44% of 86.00CYS A1061 3.00 16.67% of 18.00 ASP A1063 46.00 58.97% of 78.00 GLY A10657.00 18.92% of 37.00 LEU A1066 38.00 70.37% of 54.00 GLU A1103 5.00 6.17% of 81.00 CYS A1104 16.00 72.73% of 22.00 ARG A1105 2.00  1.80% of111.00 PRO A1106 67.00 68.37% of 98.00 CHAIN A DIFF-AREA: 858.0 (7.65%of 11223.0 total AREA for this chain)

TABLE 6 Residues of G6-23 in contact with VEGF Residues 1:211 refer tothe light chain. Residues 1001:1223 refer to the heavy chain. Residue Nrburied surface buried surface in % ASP B 28 25.00 27.17% of 92.00 SER B30 44.00 75.86% of 58.00 THR B 31 6.00 10.91% of 55.00 ALA B 32 1.0033.33% of 3.00  TYR B 49 21.00 58.33% of 36.00 PHE B 53 21.00  19.27% of109.00 TYR B 92 72.00  71.29% of 101.00 SER B1030 18.00 24.00% of 75.00ASP B1031 96.00  84.21% of 114.00 TYR B1032 28.00 48.28% of 58.00 TRPB1033 47.00 73.44% of 64.00 ILE B1051 14.00 32.56% of 43.00 PRO B105329.00 65.91% of 44.00 ALA B1054 28.00 57.14% of 49.00 GLY B1055 51.0089.47% of 57.00 GLY B1056 34.00 97.14% of 35.00 TYR B1057 37.00  21.76%of 170.00 PHE B1099 1.00 33.33% of 3.00  PHE B1101 113.00  86.92% of130.00 PHE B1102 101.00  84.17% of 120.00 LEU B1103 12.00 80.00% of15.00 PRO B1104 4.00  8.00% of 50.00 TYR B1105 54.00 68.35% of 79.00CHAIN B DIFF-AREA: 857.0 (4.38% of 19547.0 total AREA for this chain)TOTAL DIFF-AREA: 1715.0 (5.57% of 30770.0 total AREA over all chains)

(c) VEGF: Fab-12

TABLE 7 Residues of VEGF in contact with Fab-12 (PDB code 1bj1) Residues8:109 relate to monomer 1 (of the VEGF dimer). Residues 1008:1109 tomonomer 2 (of the VEGF dimer) Residue Nr buried surface buried surfacein % TYR A 45 30.00 48.39% of 62.00 LYS A 48 23.00 41.82% of 55.00 GLN A79 13.00 38.24% of 34.00 TYR A 45 30.00 48.39% of 62.00 LYS A 48 23.0041.82% of 55.00 GLN A 79 13.00 38.24% of 34.00 ILE A 80 1.00 100.00% of1.00  MET A 81 37.00 100.00% of 37.00  ARG A 82 53.00 85.48% of 62.00ILE A 83 30.00 71.43% of 42.00 LYS A 84 11.00 20.00% of 55.00 HIS A 8677.00  37.93% of 203.00 GLN A 87 119.00  80.95% of 147.00 GLY A 88 38.00100.00% of 38.00  GLN A 89 134.00 100.00% of 134.00 HIS A 90 114.00100.00% of 114.00 ILE A 91 75.00 93.75% of 80.00 GLY A 92 33.00 100.00%of 33.00  GLU A 93 83.00  62.41% of 133.00 MET A 94 5.00 50.00% of 10.00PHE A1017 25.00 55.56% of 45.00 TYR A1021 16.00 21.92% of 73.00 CHAIN ADIFF-AREA: 917.0 (8.42% of 10895.0 total AREA for this chain)

TABLE 8 Residues of Fab-12 in contact with VEGF Residues 1:211 refer tothe light chain. Residues 1001:1223 refer to the heavy chain. Residue Nrburied surface buried surface in % TYR B 91 4.00 40.00% of 10.00 SER B92 12.00 25.00% of 48.00 THR B 93 2.00 4.44% of 45.00 VAL B 94 34.0037.36% of 91.00 TRP B 96 19.00 86.36% of 22.00 THR B1030 5.00 11.36% of44.00 ASN B1031 85.00  80.19% of 106.00 TYR B1032 22.00 52.38% of 42.00GLY B1033 9.00 100.00% of 9.00  TRP B1050 53.00 91.38% of 58.00 ASNB1052 31.00 86.11% of 36.00 THR B1053 3.00 75.00% of 4.00  TYR B105493.00  51.96% of 179.00 THR B1055 6.00  7.41% of 81.00 THR B1059 29.0053.70% of 54.00 TYR B1099 12.00 100.00% of 12.00  PRO B1100 13.00 81.25%of 16.00 HIS B1101 58.00  56.31% of 103.00 TYR B1102 122.00  95.31% of128.00 TYR B1103 34.00  18.68% of 182.00 GLY B1104 41.00 50.62% of 81.00SER B1105 4.00  6.67% of 60.00 SER B1106 41.00 77.36% of 53.00 HIS B11074.00 23.53% of 17.00 TRP B1108 96.00 97.96% of 98.00 CHAIN B DIFF-AREA:832.0 (4.29% of 19409.0 total AREA for this chain) TOTAL DIFF-AREA:1749.0 (5.77% of 30304.0 total AREA over all chains)

(d) VEGF:B20

TABLE 9 Residues of VEGF buried in B20 Residue Nr buried surface buriedsurface in % LYS A 16 54.00  41.22% of 131.00 PHE A 17 43.00 100.00% of43.00  MET A 18 150.00 100.00% of 150.00 ASP A 19 55.00 90.16% of 61.00TYR A 21 75.00 94.94% of 79.00 GLN A 22 116.00  92.80% of 125.00 ARG A23 84.00  68.29% of 123.00 TYR A 25 85.00 90.43% of 94.00 CYS A 26 6.0054.55% of 11.00 HIS A 27 10.00 20.83% of 48.00 MET A 55 1.00  1.45% of69.00 CYS A 61 7.00 46.67% of 15.00 ASN A 62 3.00 12.50% of 24.00 ASP A63 4.00  5.06% of 79.00 LEU A 66 21.00 40.38% of 52.00 ASN A 100 9.0013.24% of 68.00 LYS A 101 63.00  60.00% of 105.00 GLU A 103 33.00 38.82%of 85.00 CYS A 104 13.00 54.17% of 24.00 GLU A1030 37.00 39.78% of 93.00LYS A1048 36.00 54.55% of 66.00 PRO A1053 2.00 14.29% of 14.00 GLN A10798.00 14.29% of 56.00 MET A1081 34.00 100.00% of 34.00  ILE A1083 17.0048.57% of 35.00 GLN A1089 58.00  45.67% of 127.00 ILE A1091 21.00 30.88%of 68.00 CHAIN A DIFF-AREA: 1045.0 (8.59% of 12163.0 total AREA for thischain)

TABLE 10 Residues of B20 buried in VEGF Residue Nr buried surface buriedsurface in % ARG B 30 102.00  55.43% of 184.00 ARG B 31 3.00  3.00% of100.00 SER B 32 11.00 32.35% of 34.00 TYR B 49 12.00 30.77% of 39.00 SERB 52 20.00 38.46% of 52.00 ASN B 53 41.00 82.00% of 50.00 LEU B 54 3.00 4.17% of 72.00 SER B 91 13.00 68.42% of 19.00 ASN B 92 44.00 84.62% of52.00 THR B 93 22.00 39.29% of 56.00 SER B 94 37.00 63.79% of 58.00 LEUB 96 1.00 33.33% of 3.00  TRP B1033 46.00 100.00% of 46.00  HIS B10351.00 100.00% of 1.00  TRP B1047 4.00 40.00% of 10.00 ALA B1050 2.00100.00% of 2.00  TYR B1052 61.00 81.33% of 75.00 TYR B1054 81.00  52.60%of 154.00 SER B1055 40.00 50.63% of 79.00 TYR B1057 81.00  72.32% of112.00 ASN B1059 34.00 79.07% of 43.00 TYR B1060 2.00  6.06% of 33.00TRP B1099 27.00 93.10% of 29.00 HIS B1101 118.00  70.66% of 167.00 SERB1102 16.00 20.78% of 77.00 THR B1103 4.00 28.57% of 14.00 SER B110411.00 33.33% of 33.00 PRO B1105 54.00 85.71% of 63.00 TRP B1106 52.00100.00% of 52.00  CHAIN B DIFF-AREA: 943.0 (4.90% of 19242.0 total AREAfor this chain) TOTAL DIFF-AREA: 1988.0 (6.33% of 31405.0 total AREAover all chains)

The residues having greater than 5 Å² buried surface area and/or greaterthan 5% buried were considered significantly contacted. These resultshelp describe the regions in VEGF and the regions in the antibodies thatcontact one another. Together with functional data relating to thebinding VEGF presented earlier, common features of the G6 series ofantibodies and the B20 series of antibodies can be observed. G6, G6-23and B20 antibodies, as well as others bound to both mouse and human VEGFwith relatively high affinities, unlike Fab-12 or Y0317 (data notshown). Mutations of human VEGF G88A or G88S severely affected bindingof Fab-12 or YO317 to VEGF, whereas the G6 series of antibodies and theB20 series of antibodies were relatively unaffected. Further, thebinding of antibodies such as Fab-12 to VEGF resulted in the surfacearea of G88 being 100% buried whereas the binding of G6 and G6-23resulted in G88 being less than 66% buried. Thus, it is believed thatalthough partial contact with G88 is allowable for antibodies having theproperty of recognizing both mouse and human VEGF with good affinity, itis not likely that antibodies that contact human VEGF such that thesurface area of Gly88 of human VEGF is 80% or more buried will bind toboth mouse and human VEGF with good affinity. The functional epitopemapping results also show that the footprint of the G series ofantibodies on VEGF is different from Fab-12 in that it has a greatercontact extending into the 20 s helix of VEGF (approximately residuesnumbered 10-30 of human VEGF) as well as contacting residues in the 80 sloop (approximately residues 80-94 of human VEGF). The structuralstudies correlate well with the functional studies (see FIG. 19) in thatmutations to several residues in the 20 s helix decrease binding of theG6 and B20 series antibodies. The functional studies described in FIG.19 indicate that the G6 and the B20 series antibodies interact well withresidues that are important for both Flt-1 and KDR binding as comparedto A4.6.1

Example 6 Tetranomial Diversity Libraries

To investigate whether a small subset of the natural amino acids couldbe used to generate antigen-binding surfaces, we constructed naïve heavychain phage-displayed libraries of antigen-binding fragments (Fabs)based on the humanized Fab4D5 (30), which recognizes the extracellulardomain of the human receptor tyrosine kinase ErbB2 (Fendly, B. M., etal., (1990), Cancer Res. 50:1550-1558). First, a previously describedphagemid was modified to display bivalent Fab4D5 (Fab′-zip) on thesurface of M13 bacteriophage. The gene coding for the Fab′-zip was fusedto the C-terminal domain of the M13 gene-3 minor coat protein andexpressed under the control of the phoA promoter (Lee, V., et al.,(2004) J. Immunol. Methods 284:119-132). The phagemid was modified by asingle mutation in the light chain (R66G) and by the introduction of TAAstop codons into all three heavy chain CDRs. For each libraryconstruction, the resulting phagemid (pV-0116c) was used as the “stoptemplate” in a mutagenesis reaction with oligonucleotides designed torepair simultaneously the stop codons and introduce designed mutationsat the desired sites, as described previously (Sidhu, S. S. et al.,(2004) J. Mol. Biol. 338(2):299-310; Sidhu, S S et al., (2000) Methodsin Enzymology 328:333-363).

a. Construction of KMT Library

Solvent-accessible positions within the heavy chain CDRs encoded by thephagemid were replaced by a single type of degenerate codon, KMT, thatproduced equal proportions of four amino acids (Y, A, D, S). The numberof possible tetranomial combinations of the 20 natural amino acids istoo great to be investigated exhaustively, and thus, we chosecombinations that fulfilled two criteria. Firstly, we were restricted tocombinations that could be accessed with standard DNA synthesis methods.Secondly, we ensured that each tetranomial set contained at least onesmall amino acid (glycine, serine or alanine), as we reasoned that smallresidues would provide conformational flexibility and prevent stericcrowding. A total of 18 positions were chosen for randomization:positions 28 and 30-33 in CDR-H1; positions 50, 52, 53, 54, 56 and 58 inCDR-H2; and positions 95-100a in CDR-H3. Each constructed librarycontained ˜10¹⁰ unique members, and thus, the library diversities wereonly about one order of magnitude less than the maximum theoreticaldiversity (4¹⁸=7×10¹⁰). The KMT library name corresponds to thedegenerate codon used; equimolar DNA degeneracies are represented by IUBcode (K=G/T, M=A/C, R=A/G, S=G/C, W=A/T, Y═C/T).

b. Sorting and Binding Assays for the KMT Library

Phage from the naïve heavy chain library was cycled through rounds ofbinding selection with either human vascular endothelial growth factor(hVEGF) or other antigens on 96-well Maxisorp immunoplates (NUNC) as thecapture target, as described previously (Sidhu, S. S. et al., (2004)supra; Sidhu, S S et al., (2000) Methods in Enzymology 328:333-363).Bound phage were eluted with 0.1 M HCl for 10 min and the eluant wasneutralized with 1.0 M Tris base. Phage were propagated in E. coliXL1-blue (Stratagene) with the addition of M13-KO7 helper phage (NewEngland Biolabs).

After three rounds of selection, individual clones were grown in a96-well format in 500 ul of 2YT broth supplemented with carbenicillinand M13-KO7. The culture supernatants were used in phage ELISAs todetect positive clones that bound to antigen-coated plates but not toBSA-coated plates (Sidhu, S. S. et al., (2004) supra). Positive cloneswere subjected to DNA sequence analysis and assessed forantigen-specific binding with phage enzyme-linked immunosorbant assays(ELISAs) (Sidhu, S. S. et al., (2004) supra). Approximately 100 cloneswere screened against each antigen and specific binding clones wereidentified in each case. DNA sequencing revealed the number of uniqueclones isolated against each antigen. At least one tyrosine-containinglibrary was successful against each antigen. In particular, Library-KMTwas successful against 3 of the 4 antigens and generated 11 uniqueclones against human vascular endothelial growth factor (hVEGF).

c. Second KMT Library—Light Chain Diversity

We constructed new versions of the KMT Library in which the CDR-H1 andCDR-H2 diversities were the same as described above, but the diversityof CDR-H3 was increased by allowing for all possible length variationsranging from 3-15 residues inserted between residues 94 and 100b. Alltogether, the pooled libraries contained a diversity of ˜10¹⁰ uniquemembers that were cycled through selections for binding to hVEGF. PhageELISA screens identified 93 hVEGF binders and DNA sequencing revealed 15unique sequences (FIG. 36). Most of the clones contained CDR-H3sequences with seven inserted residues, but we also identified twoclones that contained longer insertions. The unique clones weresubjected to competitive phage ELISAs (Sidhu, S. S. et al., (2004)supra) and exhibited estimated affinities in the 10 micromolar range(data not shown).

d. Affinity Maturation of Unique Clones from Second KMT Library

We next investigated whether the low affinity anti-VEGF clones could beaffinity matured to obtain Fabs with affinities comparable to those ofnatural antibodies. To this end, we recombined the 15 heavy chains (FIG.36) with a light chain library in which 12 solvent-accessible positionswere replaced with the same tetranomial KMT codon. Specifically, thefollowing light chain positions were randomized: positions 28-32 inCDR-L1, positions 50 and 53 in CDR-L2, and positions 91-94 and 96 inCDR-L3. The libraries contained ˜10¹⁰ unique members which greatlyexceeded the theoretical diversity of possible light chains (4¹²=2×10⁷).The phagemid selected for the display of a heavy chain sequence andabove light chain sequence had been modified by the introduction of TAAstop codons into all three light chain CDRs. The resulting phagemid wasused as the “stop template” in a mutagenesis reaction that repaired thestop codons and introduced desired mutations, as described above.

Phage from the light chain libraries were incubated for 2 h at roomtemperature in PBS, 0.05% Tween 20 (Sigma), 0.5% Superblock (Pierce)with 100 nM hVEGF biotinylated with Sulfo-NHS-LC-Biotin reagent(Pierce). Biotinylated hVEGF and bound phage were captured for 5 minwith neutravidin (Pierce) immobilized on Maxisorp immunoplates. Theplates were washed with PBS, 0.05% Tween 20 and the bound phage wereeluted and propagated for additional rounds of selection, as describedabove.

After selection, individual clones were grown in a 96-well format in 500ul of 2YT broth supplemented with carbenicillin and M13-KO7. The culturesupernatants were used in phage ELISAs to detect positive clones thatbound to antigen-coated plates but not to BSA-coated plates (Sidhu, S.S. et al., (2004) supra). Positive clones were subjected to DNA sequenceanalysis.

hVEGF in solution was used for a high stringency selection. We sequenced256 clones and identified 64 unique light chains combined with 9 of the15 heavy chains (top 9 sequences in FIG. 36). Competitive phage ELISAswere used to estimate affinities of clones (Sidhu, S. S. et al., (2004)supra). Such ELISAs were carried out generally as follows. Phage cloneswere propagated from a single colony by growing in 40 ml of 2YT culturesupplemented with carbenicillin and KO7 helper phage overnight at 30° C.Phage purified by PEG/NaCl precipitation were first serially diluted inPBST and tested for binding to an antigen-coated plate (hVEGF or mVEGF).The dilution that gave 50-70% saturating signal was used in the solutionbinding assay in which phage were first incubated with increasingconcentration of antigen for 1-2 h and then transferred to antigencoated plate to capture the unbound phage for 10-15 min. An IC₅₀ wascalculated as the concentration of antigen in solution binding stagethat inhibited 50% of the phage from binding to immobilized antigen. Thethree highest affinity phage clones were YADS1, YADS2 and YADS3, whichclones were converted into Fabs.

e. YADS1, 2 and 3 Fab Binding Affinities

YADS1, 2 and 3 were purified as free Fab proteins for detailed analysis.The sequence of these Fabs are provided below. See also FIG. 39.

YADS1 Light Chain (SEQ ID NO: 19)DIQMTQSPSSLSASVGDRVTITCRASQASYSSVAWYQQKPGKAPKLLIYAASYLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSSASPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECYADS1 Heavy Chain (SEQ ID NO: 20)EVQLVESGGGLVQPGGSLRLSCAASGFDIYDDDIHWVRQAPGKGLEWVAYIAPSYGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRSSDASYSYSAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH YADS2 Light Chain (SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQSYAYAVAWYQQKPGKAPKLLIYDASYLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSSPDTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECYADS2 Heavy Chain (SEQ ID NO: 22)MKKNIAFLLASMFVFSIATNAYAEVQLVESGGGLVQPGGSLRLSCAASGFAISDYDIHWVRQAPGKGLEWVADIAPYAGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRSSYAYYAAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHYADS3 Light Chain (SEQ ID NO: 23)DIQMTQSPSSLSASVGDRVTITCRASQASYYDVAWYQQKPGKAPKLLIYAASYLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYAPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECYADS3 Heavy Chain (SEQ ID NO: 24)MKKNIAFLLASMFVFSIATNAYAEVQLVESGGGLVQPGGSLRLSCAASGFSISDYDIHWVRQAPGKGLEWVAAIAPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRSSYAYYSAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTH

Fab proteins were purified from E. coli shake-flask cultures, asdescribed previously (Muller, Y A et al., (1998) Structure 6:1153-1167).Generally, the variable domains were cloned into vectors designed forFab expression in E. coli or transient IgG expression in mammaliancells. The Fab expression vector was derived from the phage displayphagemid by deleting the sequence encoding for cP3 and adding aterminator sequence (GCTCGGTTGCCGCCGGGCGTTTTTTAT) (SEQ ID NO:920) about20 nucleotides downstream from the stop codon at the end of C_(H)1. Fabprotein was generated by growing the transformed 34B8 E. coli cells inAP5 media at 30° C. for 24 h as described (Presta, L. G., et al., (1997)Cancer Res. 57, 4593-4599). Fab was purified with Protein G affinitychromatography. The production yield for Fab was typically 5-10 mg/L insmall scale shake flask growth and 0.5-3 g/L in fermenter growth.

Binding kinetic values for the purified Fabs based on surface plasmonresonance are shown below. hVEGF₈₋₁₀₉ or mVEGF were immobilized on CM5chips at ˜100 response units in a BIAcore™-3000, as described previously(Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881). Serial dilutionsof Fab proteins (3-500 nM) were injected, and binding responses on thehVEGF or mVEGF flow cell were corrected by subtraction of responses on ablank flow cell. For kinetic analysis, a 1:1 Languir model with separatefittings of k_(on) and k_(off) was used. The K_(d) values were estimatedfrom the ratios of k_(on) and k_(off). All three Fabs bound with highaffinity to hVEGF, but only two exhibited appreciable affinity for thehighly homologous murine VEGF (mVEGF, 90% amino acid identity). Wereasoned that YADS2 and YADS3 recognized VEGF through a very similarmechanism, as they exhibited high sequence homology in their CDRs andbound to both human and murine VEGF. In contrast, YADS1 likelyrepresented a unique mode of antigen recognition as it contained verydifferent CDR sequences and did not recognize mVEGF.

Kinetic analysis of Fabs binding to immobilized VEGF k_(on) (s⁻¹ M⁻¹)k_(off) (s⁻¹) K_(d) (nM) Fab hVEGF mVEGF hVEGF mVEGF hVEGF mVEGF YADS 13 × 10⁵ ND¹ 5 × 10⁻⁴ ND¹ 1.8 ± 0.3 >1000 YADS 2 1 × 10⁶ 8 × 10⁵ 1 × 10⁻²4 × 10⁻³ 10 ± 2  5.0 ± 0.8 YADS 3 1 × 10⁶ 2 × 10⁶ 3 × 10⁻³ 5 × 10⁻³ 2.0± 0.4 3.7 ± 0.6 ¹Values could not be determined due to the low affinityof the interaction.

YADS2 Fab inhibited VEGF induced HUVEC proliferation (FIG. 44).Generally, the HUVEC proliferation assay was performed as described byothers previously (Chen, Y., (1999) J. Mol. Biol. 293:865-881).Basically, HUVEC assays are carried out by testing YADS2 (or G6, etc.)for their ability to block either human or murine VEGF from stimulatingthe growth of human umbilical vein endothelial cells (HuVECs).Approximately 3000 HuVECs were plated in each well of the 96-well cellculture plate and fasted in F12:DMEM 50:50 medium supplemented with 1.5%(v/v) filtered fetal bovine serum for over night. Fresh assay mediumwith fixed amounts of VEGF (0.6 nM final concentration), determined byfirst titrating VEGF that can stimulate submaximal DNA synthesis, andincreasing concentrations of anti-VEGF Fab were then added to the cells.After incubation at 37 C for 24 h, the cells were pulsed with 0.5mu.Ci/well of [3H] thymidine for 24 h and harvested for counting with aTopCount Microplate Scintillation counter. The IC50 was determined to beapproximately 12 nM.

Example 7 Crystallization, Structure, Determination and Refinement ofYADS1 and YADS2

We wanted to study the structural basis for antigen recognition, and so,the crystal structures of YADS1 and YADS2 in complex with hVEGF.

a. Fab Protein Preparation for Crystal Structure Analysis

Whole cell broth was obtained from a 10 liter E. coli fermentation. Thecells were lysed with a Manton-Gaulin homogenizer. The suspension wascentrifuged, the supernatant was loaded on a protein A-Sepharose column(Genentech, Inc.), and the column was eluted with 0.1 M acetic acid. ThepH was adjusted to 4.0 with 1.0 M Tris, pH 8.0 and the eluant was loadedon a SP-Sepharose column (Pharmacia). The column was washed withequilibration buffer (20 mM MES, pH 5.5) and Fab protein was eluted witha NaCl gradient in equilibration buffer. Residues 8-109 of human VEGFwere expressed, refolded, and purified as previously described(Christinger, H. W., et al., (1996). Prot. Struct. Funct. Genet. 26,353-357).

The complex between each Fab and the receptor-binding fragment of hVEGFwas formed and purified, as described previously (Muller, Y. A., (1998),supra). The complex (in PBS, 25 mM EDTA) was concentrated to an opticaldensity of A₂₈₀=10. Hanging-drop experiments were performed using thevapor-diffusion method with 10 ul drops consisting of a 1:1 ratio ofprotein solution and reservoir solution. The reservoir solution for theYADS1 complex was 0.2 M ammonium sulfate, 25% PEG 3350 (w/v), 0.1 MHEPES, pH 7.5. The reservoir solution for the YADS2 complex was 1.0 Mlithium chloride, 10% PEG 6000 (w/v), 0.1 M MES, pH 6.0. After 1-2 weeksat 19° C., plate or spindle shaped crystals grew for the YADS1 or YADS2complex, respectively.

Crystals were incubated in reservoir solution supplemented with 25%glycerol prior to flash freezing. A data set was collected from a singlefrozen crystal at the beam line 5.0.2 of the Advanced Light Source(Berkeley) for YADS1 and at the beam line 9.2 of the StanfordSynchrotron Radiation Laboratory (Stanford University) for YADS2. Thedata was processed using the programs DENZO and SCALEPACK (Otwinowski,Z. M., (1997) Methods Enzymol. 276:307-326). The structures were solvedby molecular replacement using the program AMoRe (CCP4 (1994) ActaCryst. D50:760-763) and the coordinates of a previously solved Fab-hVEGFcomplex (PDB entry 1BJ1). The structure was refined using the programsREFMAC (CCP4 (1994), supra). The models were manually adjusted usingprogram O (Jomes, T. A., et al., (1991) Acta Crystallogra A 47(Pt2):969-995). The following programs were used to calculate thesurface areas of interaction RESAREA version 3.2: 05/08/93 and AREAIMOLversion 3.2: 19/12/95. These programs are part of the CCP4 suiteCollaborative Computational Project, Number 4. 1994 (“The CCP4 Suite:Programs for Protein Crystallography” Acta Cryst. D50, 760-763). Thecrystal structures of YADS1 and YADS2 in complex with hVEGF were solvedand refined at 2.65 and 2.8 Å resolution, respectively (Table 11).

TABLE 11 Data collection and refinement statistics for YADS1 and YADS2hVEGF complexes. YADS 1 YADS 2 A. Unit cell Space group P2₁ C222₁ a (Å)83.3 96.5 b (Å) 112.5 149.6 c (Å) 105.8 117.4 Beta (deg.) 105.8 B.Diffraction Data Resolution (Å)  50-2.6 (2.7-2.6)^(a)  50-2.8(2.9-2.8)^(a) No. of reflections 156868 111731 No. of unique 41828 20861reflections R_(merge) ^(b) 0.066 (0.356)^(a) 0.076 (0.399)^(a)Completeness (%) 99.9 (99.1)^(a) 97.3 (83.2)^(a) C. Refinement R_(work)^(c,) R_(free) ^(c) 0.212, 0.271 0.218, 0.254 No. of non-H 8104 4077atoms No. of waters 110 0 rmsd bond length 0.011 0.011 (Å) rmsd angles1.2 1.3 (deg.) ^(a)Values for the outer resolution shell are given inparantheses. ^(b)R_(merge) = Σ_(hkl)|I_(hkl) −<I_(hkl)>|)/Σ_(hkl)<I_(hkl)>, where I_(hkl) is the intensity ofreflection hkl, and <I_(hkl)> is the average intensity of multipleobservations. ^(c)R_(work) = Σ|F_(o) − F_(c)|/ΣF_(o), where F_(o) andF_(c) are the observed and calculated structure factor amplitudes,respectively. R_(free) is the R factor for a randomly selected 5% ofreflections which were not used in the refinement

The surface area of each residue of an anti-VEGF antibody that is buriedin VEGF (Å²) is reported below together with the percentage of the totalsurface area of the residue that is buried. Also reported is the surfacearea of each residue of VEGF antibody that is buried in VEGF (Å²) isreported below together with the percentage of the total surface area ofthe residue that is buried. See values for YADS-1:VEGF and YADS-2:VEGFcomplexes below. In all cases, because VEGF is a dimer, the residuenumbers of VEGF referring to monomer 1 (of the VEGF dimer) are 8-109 andresidue numbers of VEGF referring to monomer 2 (of the VEGF dimer) are1008-1109. The first column in each table below recites the residuenumbers of the protein being examined (either VEGF or an anti-VEGFantibody) (e.g., for section (a) below, TYR A 45 refers to Y45, LYSA1016 refers to K16 of VEGF). The second column recites the buriedsurface of that residue (Å²). The third column recites the buriedsurface for that residue as a percentage of the surface area of thewhole residue

(a) VEGF:YADS-1

TABLE 12 Residues of VEGF in contact with YADS-1 Residue Nr buriedsurface buried surface in % TYR A 45 8.00 20.51% of 39.00 ILE A 46 36.0037.89% of 95.00 LYS A 48 14.00 20.90% of 67.00 GLN A 79 31.00 81.58% of38.00 MET A 81 24.00 75.00% of 32.00 ARG A 82 7.00 14.58% of 48.00 ILE A83 33.00 76.74% of 43.00 LYS A 84 25.00 40.98% of 61.00 PRO A 85 8.0014.55% of 55.00 HIS A 86 107.00  54.04% of 198.00 GLN A 87 110.00 71.43% of 154.00 GLY A 88 38.00 95.00% of 40.00 GLN A 89 103.00  88.79%of 116.00 HIS A 90 93.00  75.61% of 123.00 ILE A 91 71.00 89.87% of79.00 GLY A 92 12.00 60.00% of 20.00 GLU A 93 21.00  17.50% of 120.00LYS A1016 31.00  25.20% of 123.00 PHE A1017 21.00 46.67% of 45.00 META1018 14.00  10.14% of 138.00 ASN A1062 10.00 28.57% of 35.00 ASP A10636.00  6.38% of 94.00 GLU A1064 89.00  49.17% of 181.00 LYS A1107 0.00 0.00% of 165.00 CHAIN A DIFF-AREA: 912.0 (8.16% of 11170.0 total AREAfor this chain) TOTAL DIFF-AREA: 912.0 (8.16% of 11170.0 total AREA overall chains

(b) VEGF: YADS-2

TABLE 13 Residues of VEGF in contact with YADS-2 Residue Nr buriedsurface buried surface in % ILE A 46 6.00  8.82% of 68.00 LYS A 48 39.0060.94% of 64.00 GLN A 79 17.00 45.95% of 37.00 MET A 81 29.00 96.67% of30.00 ILE A 83 32.00 86.49% of 37.00 PRO A 85 22.00 38.60% of 57.00 HISA 86 128.00  64.00% of 200.00 GLN A 87 32.00  23.88% of 134.00 GLY A 8822.00 64.71% of 34.00 GLN A 89 119.00  88.15% of 135.00 HIS A 90 20.0021.74% of 92.00 ILE A 91 55.00 67.07% of 82.00 LYS A1016 1.00  0.87% of115.00 PHE A1017 45.00 90.00% of 50.00 MET A1018 47.00  40.52% of 116.00TYR A1021 7.00  8.97% of 78.00 ASP A1063 27.00 36.49% of 74.00 GLY A10656.00 13.33% of 45.00 LEU A1066 32.00 55.17% of 58.00 CYS A1104 3.0012.50% of 24.00 ARG A1105 7.00  5.65% of 124.00 PRO A1106 29.00 37.66%of 77.00 CHAIN A DIFF-AREA: 725.0 (6.17% of 11744.0 total AREA for thischain). TOTAL DIFF-AREA: 725.0 (6.17% of 11744.0 total AREA over allchains)

The residues having greater than 5 Å² buried surface area and/or greaterthan 5% buried were considered significantly contacted. These resultshelp describe the regions in VEGF and the regions in the antibodies thatcontact one another. Together with functional data relating to thebinding VEGF presented earlier, common features of the G6 series ofantibodies YADS-2 and YADS-3 antibodies can be observed. The binding ofantibodies such as Fab-12 and YADS-1 to VEGF resulted in the surfacearea of G88 being 100% and 95%, respectively, buried whereas the bindingof G6, G6-23 and YADS-2 resulted in G88 being only 66% or less buried.

The Fab frameworks were essentially unchanged in comparison with thestructure of the parental Fab4D5; the C_(α) atoms of the YADS1 and YADS2frameworks superimposed with Fab4D5 with root mean square deviations(rmsd) of 0.87 and 0.55 Å, respectively. The Cα atoms of the hVEGFmolecules in the two structures superimpose well onto each other withrmsds of 0.7 Å for 87 Cα positions. The largest deviation of 3.7 Åoccurs at residue glutamic acid 64. The loop containing this residue hasinherent flexibility as shown by Muller et al., supra.

In both complexes, antigen recognition was entirely mediated by contactswith the CDR loops. In terms of buried surface area, YADS1 used both theheavy (498 Å²) and light chain (407 Å²), whereas YADS2 used mostly theheavy chain (543 Å²) and a small contribution from the light chain (157Å²). Notably, residues at randomized positions accounted for essentiallyall of the buried surface area (98% and 100% for YADS1 and YADS2,respectively), and furthermore, the buried surface area involved almostentirely side chain atoms (82% and 80% for YADS1 and YADS2,respectively). Thus, both Fabs bound to antigen through interactionsthat were almost entirely mediated by side chains located at positionsthat were randomized in the libraries.

On the hVEGF side, the structural epitopes for binding to YADS1 andYADS2 overlap with each other, and also, with the structural epitope forbinding to domain 2 of the hVEGF receptor Flt-1 (Flt-1_(D2),). The YADS1and YADS2 antibodies can inhibit binding of Flt to human VEGF in vitro(data not shown), and they are expected to inhibit binding of KDR tohuman VEGF too. Nonetheless, there are significant differences betweenthe structural epitopes for the two Fabs. In particular, of the 11residues that differ between human and murine VEGF, only residue 88 isin contact with the Fabs, but the interactions involving this residueexplain the differing affinities of YADS1 and YADS2 for mVEGF. In theYADS2 complex, Gly88 is partially exposed to solvent, while in the YADS1complex it is completely buried in the interface. Murine VEGF contains alarger serine residue at position 88; this substitution can be readilyaccommodated in the YADS2 complex, but in the YADS1 model, theintroduction of a serine side chain at the buried Gly88 position wouldrequire major rearrangements for the complex to be preserved.

As described above, both Fabs bind to antigen through contacts almostexclusively involving side chains at varied sites. In total, the CDRs ofYADS1 and YADS2 contain 66 residues derived from randomized codons, andthese residues are almost equally distributed amongst the four aminoacid types allowed in the library design. However, when we consider thesubset of residues that make contact with antigen, there is a clear biasin that 16 tyrosines account for 50% of the contact residues. Indeed,all but two of the tyrosines selected in the CDRs of YADS1 and YADS2make contact with antigen, and all told, tyrosines contribute 71% of thesurface area buried upon complexation with hVEGF. Thus, essentiallyevery selected tyrosine side chain is involved in directly mediatingantigen recognition and the other selected amino acids apparently playauxiliary roles.

Despite the predominance of tyrosine in the synthetic antigen-bindingsites, an examination of the heavy atom (non-hydrogen) content of buriedsurface areas reveals that the Fab-hVEGF interfaces are no morehydrophobic than the interface between hVEGF and Flt-1_(D2). On thehVEGF side, the heavy atom composition of the buried surface area isvery similar in all three cases, being composed predominantly of carbonbut also containing significant proportions of nitrogen and oxygen.Within the buried surface areas of the Fabs, nitrogen atoms are almostentirely absent, because the side chains allowed in the libraries werecomposed entirely of carbon, oxygen and hydrogen. Nonetheless, both Fabsbury a large number of oxygen atoms upon binding to hVEGF, and in bothcases, the proportion of the buried surface area contributed by carbonis considerably less than that contributed by carbon to the buriedsurface area of Flt-1_(D2). Thus, the predominance of tyrosine in thesynthetic CDRs does not produce highly hydrophobic Fab-antigeninterfaces dominated by aromatic interactions. On the contrary, thetyrosine residues make specific contacts with a wide variety of residueson the hVEGF surface, and these interactions utilize both the side chainhydroxyl groups and aromatic rings.

Thus, we circumvented the complexity of the natural immune system byusing precisely defined synthetic libraries, and as a result, we wereable to investigate the special role that tyrosine plays inantigen-binding sites. We generated libraries with restricteddiversities and displayed the diverse surfaces on a fixed scaffoldformed by the framework regions and buried CDR residues. Our resultsdramatically demonstrate that, in the context of a suitable scaffold,the tyrosine side chain is capable of mediating most of the contactsnecessary for high affinity antigen recognition. Thus, it seems verylikely that the overabundance of tyrosine in natural antigen-bindingsites is a consequence of the side chain being particularly well suitedfor making productive contacts with antigen.

This supposition is also consistent with the chemical nature oftyrosine. As noted previously, the tyrosine side chain is large enoughto sweep out large volumes of space with only a few torsion angles, andit can form hydrogen bonds, hydrophobic interactions and attractiveelectrostatic interactions with positively charged groups (Zemlin, M.,et al., (2003) J. Mol. Biol. 334:733-749). In addition, the unchargedtyrosine side chain avoids electrostatic repulsion effects, and itsmidrange hydrophilicity allows it to adapt favorably to both hydrophilicand hydrophobic environments (Zemlin, (2003), supra; Ivanov, I., et al.,(2002) in The Antibodies, eds. Zanetti, M. & Capra, J. (Taylor & Fancis,London, New York), pp. 43-67; Mian, I. S., et al., (1991) J. Mol. Biol.217:133-151).

We also observed that, while alanine and serine residues did not makemany direct contacts with antigen, they allowed for space andconformational flexibility which may be crucial for appropriatepositioning of the large tyrosine side chains. Thus, these smallresidues may serve an auxiliary function in facilitating productivecontacts between tyrosine and antigen. It is worth noting that, perhapsnot coincidentally, serine is also highly abundant in naturalantigen-binding sites (Mian, (1991), supra). Finally, the paucity ofantigen contacts mediated by aspartate suggests that it may be possibleto further minimize the chemical diversity of these syntheticantigen-binding sites.

Example 8 Anti-VEGF Antibodies from YADS-A and YADS-B Libraries

(a) Construction of Phage-Displayed Fab Libraries YADS-A and YADS-B

Two phage displayed libraries (YADS-A and YADS-B) were constructed, asgenerally described in Example 6, with a previously described phagemiddesigned to display bivalent Fab moieties dimerized by a leucine zipperdomain inserted between the Fab heavy chain and the C-terminal domain ofthe gene-3 minor coat protein (P3C), except that the following positionsof 4D5 were randomized as follows:

Randomized Positions Library CDRH1 CDRH2 CDRH3 YADS-A 28, 30, 31, 32, 3350, 52, 53, 54, 56, 95, 96, 97, 98, 99, 58 100, 100a YADS-B 28, 30, 31,32, 33 50, 52, 53, 54, 56, 95, 96, 97, 98, 99, 58 100, 100a

For library YADS-A, two separate mutagenesis reactions were performed.In the first reaction, diversity was introduced into CDR-H1, CDR H2 andCDR-H3 with oligonucleotides YADS-H1, YADS-H2 and YADS-H3-7,respectively. This resulted in the introduction of degenerate codonsthat encoded for the four amino acids tyrosine, alanine, aspartate, andserine (YADS). In the second reaction, diversity was introduced intoCDR-H1, CDR H2 and CDR-H3 with oligonucleotides YTNS-H1, YTNS-H2 andYTNS-H3-7, respectively. This resulted in the introduction of degeneratecodons that encoded for the four amino acids tyrosine, threonine,asparagine, and serine (YTNS). The two reactions were pooled.

For library YADS-B, 13 separate mutagenesis reactions were performed.The reactions resulted in the introduction of degenerate codons thatencoded for the four amino acids tyrosine, alanine, aspartate, andserine (YADS). In each reaction, diversity was introduced into CDR-H1and CDR-H2 with oligonucleotides YADS-H1 and YADS-H2. For each reaction,one of the following oligonucleotides was used to introduce diversityinto CDR-H3: YADS-H3-3, YADS-H3-4, YADS-H3-5, YADS-H3-6, YADS-H3-7,YADS-H3-8, YADS-H3-9, YADS-H3-10, YADS-H3-11, YADS-H3-12, YADS-H3-13,YADS-H3-14, or YADS-H3-15. The 13 reactions were pooled.

For both libraries, the pooled mutagenesis reactions were electroporatedin E. coli SS320 (Sidhu et al., supra). The transformed cells were grownovernight in the presence of M13-KO7 helper phage (New England Biolabs,Beverly, Mass.) to produce phage particles that encapsulated thephagemid DNA and displayed Fab fragments on their surfaces. The size oflibraries YADS-A and YADS-B were both 7×10⁹.

(b) Selection of Anti-hVEGF Specific Antibodies from YADS-A and YADS-BNaïve Libraries.

Phage from library YADS-A and YADS-B were cycled seperately throughrounds of binding selection to enrich for clones binding to h-VEGF. Thebinding selections were conducted using previously described methods(Sidhu et al., supra).

NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. withcapture target (5 μg/mL) and blocked for 2 h with BSA (Sigma). Afterovernight growth at 37° C., phage were concentrated by precipitationwith PEG/NaCl and resuspended in PBS, 0.5% BSA, 0.05% Tween 20 (Sigma),as described previously (Sidhu et al., supra). Phage solutions (˜10¹²phage/mL) were added to the coated immunoplates. Following a 2 hincubation to allow for phage binding, the plates were washed 10 timeswith PBS, 0.05% Tween20. Bound phages were eluted with 0.1 M HCl for 10min and the eluant was neutralized with 1.0 M Tris base. Eluted phagewere amplified in E. coli XL1-blue and used for further rounds ofselection.

The libraries were subjected to 4 rounds of selection against eachtarget protein. Individual clones from each round were grown in a96-well format in 500 μL of 2YT broth supplemented with carbenicillinand M13-VCS, and the culture supernatants were used directly in phageELISAs (Sidhu et al., supra) to detect phage-displayed Fabs that boundto plates coated with target protein but not to plates coated with BSA.A clone was considered to be a specific binder if the ELISA signal ontarget coated plates was at least 20 times greater than that on BSAcoated plates.

Specific binders were sequenced, and the sequences of unique clones areshown in FIGS. 37 and 38 for libraries YADS-A and YADS-B, respectively.Sequences from FIG. 37 were obtained by sorting with human VEGF8-109.Sequences from FIG. 38 were obtained by sorting with murine VEGF.

Example 9 NNK Variants of YADS2

(a) Construction of phage-displayed Fab libraries by randomizingselected positions of the YADS2 anti-VEGF antibody with the NNK codon.

Phage-displayed Fab libraries were constructed using a phagemid vectorthat resulted in the display of bivalent Fab moieties dimerized by aleucine zipper domain inserted between the Fab heavy chain and theC-terminal domain of the gene-3 minor coat protein (P3C). This vectorcomprised the YADS2 sequence. The humanized antibody YADS2 variabledomains were expressed under the control of the IPTG-inducible Ptacpromoter.

Library NNK was constructed with randomized residues in the heavy chainCDR-3 of YADS2. The specific residues that were randomized are 50, 95,97, 99, 100, and 100a of the heavy chain.

At each of the randomized positions, the wild-type codon was replaced bya degenerate NNK codon (N=A/T/G/C, K=G/T in an equimolar ratio) thatencoded for all 20 natural amino acids.

Libraries were constructed using the method of Kunkel (Kunkel, T. A.,Roberts, J. D. & Zakour, R. A., Methods Enzymol. (1987), 154, 367-382)with previously described methods (Sidhu, S. S., Lowman, H. B.,Cunningham, B. C. & Wells, J. A., Methods Enzymol. (2000), 328,333-363). A unique “stop template” version of the Fab display vector wasused to generate the NNK library. We used a template phagemid bearingthe gene coding for YADS2 fab with TAA stop codons inserted at positions30, 33, 52, 54, 56, 57, 60, 102, 103, 104, 107, and 108 of the heavychain. Mutagenic oligonucleotides with degenerate NNK codons at thepositions to be diversified were used to simultaneously introduce CDRdiversity and repair the stop codons. Diversity was introduced intoCDR-H3 with the oligonucleotide named NNK-H1 (GCA GCT TCT GGC TTC GCTATT TAT GAT TAT GAT ATA CAC TGG GTG CGT (SEQ ID NO:25)), NNK-H2 (CTG GAATGG GTT GCA NNK ATT GCT CCA TAT GCT GGT GCT ACT GCT TAT GCC GAT AGC GTC(SEQ ID NO:26)) and NNK-H3 GTC TAT TAT TGT AGC CGC NNK TCT NNK GCT NNKNNK NNK GCT ATG GAC TAC TGG (SEQ ID NO:27)). The mutagenicoligonucleotides for all three heavy chain CDRs were incorporatedsimultaneously in a single mutagenesis reaction, so that simultaneousincorporation of the mutagenic oligonucleotide resulted in theintroduction of the designed diversity at each position andsimultaneously repaired all the TAA stop codons, thus generating an openreading frame that encoded a Fab library member fused to ahomodimerizing leucine zipper and P3C. Note that the oligonucleotideNNK-H1 does not contain any degenerate codons and is added to themutagenesis reaction to repair the TAA stop codons and introduce thewild type YADS2 sequence.

The mutagenesis reactions were electro porated into E. coli SS320 (Sidhuet al., supra), and the transformed cells were grown overnight in thepresence of M13-KO7 helper phage (New England Biolabs, Beverly, Mass.)to produce phage particles that encapsulated the phagemid DNA anddisplayed Fab fragments on their surfaces. Each library containedgreater than 5×10⁹ unique members.

(b) Selection of Specific Anti-VEGF Antibodies from the NNK Library

Phage from library NNK were cycled through rounds of binding selectionto enrich for clones binding to human VEGF. The binding selections wereconducted using previously described methods (Sidhu et al., supra).

NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. withcapture target (5 μg/mL) and blocked for 2 h with Superblock TBS(tris-buffered saline) (Pierce). After overnight growth at 37° C., phagewere concentrated by precipitation with PEG/NaCl and resuspended inSuperblock TBS, 0.05% Tween 20 (Sigma), as described previously (Sidhuet al., supra). Phage solutions (˜10¹² phage/mL) were added to thecoated immunoplates. Following a 2 h incubation to allow for phagebinding, the plates were washed 10 times with PBS, 0.05% Tween 20. Boundphage were eluted with 0.1 M HCl for 10 min and the eluant wasneutralized with 1.0 M Tris base. Eluted phage were amplified in E. coliXL1-blue and used for further rounds of selection.

The libraries were subjected to 5 rounds of selection against VEGF.Individual clones from each round of selection were grown in a 96-wellformat in 500 μL of 2YT broth supplemented with carbenicillin andM13-VCS, and the culture supernatants were used directly in phage ELISAs(Sidhu et al., supra) to detect phage-displayed Fabs that bound toplates coated with target protein but not to plates coated with BSA.Specific binders were defined as those phage clones that exhibited anELISA signal at least 15-fold greater on target-coated plates incomparison with BSA-coated plates. Individual clones were screened after3 to 5 rounds of selection for VEGF binding.

Individual clones representing specific binders were subjected to DNAsequence analysis, and the sequences of the randomized CDR positions areshown in FIG. 40. The affinity of the different YADS variants wasestimated by using a “two point competitive phage ELISA”. The three bestclones were produced as soluble Fab and were tested for their affinitywith respect to hVEGF. BIAcore data was obtained according to Chen etal., J Mol Biol. (1999), 293(4):865-81. Briefly, binding affinities werecalculated from association and dissociation rate constants measuredusing a BIAcore™-3000 surface plasmon resonance system (BIAcore, Inc.,Piscataway, N.J.). A biosensor chip was activated for covalent couplingof VEGF using N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's (BIAcore, Inc., Piscataway, N.J.) instructions. hVEGF ormVEGF was buffer-exchanged into 10 mM sodium acetate, pH 4.8 and dilutedto approximately 30 mg/ml. Aliquots of VEGF were injected at a flow rateof 2 microL/minute to achieve approximately 200-300 response units (RU)of coupled protein. A solution of 1 M ethanolamine was injected as ablocking agent. For kinetics measurements, twofold serial dilutions ofFab were injected in PBS/Tween buffer (0.05% Tween20 inphosphate-buffered saline) at 25° C. at a flow rate of 10 microL/minute.Equilibrium dissociation constants, Kd values from surface plasmonresonance measurements were calculated as k_(off)/k_(on). The BIAcore™data is summarized as follows:

Clone Name hVEGF (nM) mVEGF (nM) NNK-1 0.60 0.24 NNK-2 2.0 13 NNK-3 6.0

Example 10 Binomial Diversity Libraries

(a) Construction of Phage-Displayed Fab Libraries with CDR ResiduesRandomized as Only Tyr or Ser

Phage-displayed Fab libraries were constructed using a phagemid vectorthat resulted in the display of bivalent Fab moieties dimerized by aleucine zipper domain inserted between the Fab heavy chain and theC-terminal domain of the gene-3 minor coat protein (P3C). This vectorcomprises the humanized antibody 4D5 variable domains under the controlof the IPTG-inducible Ptac promoter as described above. The humanizedantibody 4D5 is an antibody which has mostly human consensus sequenceframework regions in the heavy and light chains, and CDR regions from amouse monoclonal antibody specific for Her-2. The method of making theanti-Her-2 antibody and the identity of the variable domain sequencesare provided in U.S. Pat. Nos. 5,821,337 and 6,054,297.

Two libraries were constructed. Library YS-A was constructed withrandomized residues in all three heavy chain CDRs, while Library YS-Bwas constructed with randomized residues in all three heavy chain CDRsand light chain CDR3. The specific residues that were randomized areshown below.

Randomized Positions Library CDRL3 CDRH1 CDRH2 CDRH3 YS-A 28, 30, 31,50, 52, 53, 95, 96, 97, 98, 99, 32, 33 54, 56, 58 100, 100a YS-B 91-94,96 28, 30, 31, 50, 52, 53, 95, 96, 97, 98, 99, 32, 33 54, 56, 58 100,100a

At each of the randomized positions, the wild-type codon was replaced bya degenerate TMT codon (M=A/C in an equimolar ratio) that encoded forTyr and Ser in an equimolar ratio. In addition, the length of CDRH3 wasvaried by using oligonucleotides that replaced the 7 wild-type codonsbetween positions 101 to 107 with varying numbers of TMT codons (7 to 20for Library YS-A and 7 to 15 for Library YS-B). In addition, the CDRL3of Library YS-B was randomized so that 50% of the library memberscontained a deletion at position number 91 while the other 50% containedthe wildtype Gln residue at this position.

Libraries were constructed using the method of Kunkel (Kunkel, T. A.,Roberts, J. D. & Zakour, R. A., Methods Enzymol. (1987), 154, 367-382)with previously described methods (Sidhu, S. S., Lowman, H. B.,Cunningham, B. C. & Wells, J. A., Methods Enzymol. (2000), 328,333-363). A unique “stop template” version of the Fab display vector wasused to generate both libraries YS-A and YS-B. We used a templatephagemid designated pV0350-4 with TAA stop codons inserted at positions30, 33, 52, 54, 56, 57, 60, 102, 103, 104, 107, and 108 of the heavychain. No stops were introduced in the light chain CDR3. Mutagenicoligonucleotides with degenerate TMT codons at the positions to bediversified were used to simultaneously introduce CDR diversity andrepair the stop codons. For both libraries, diversity was introducedinto CDR-H1 and CDR-H2 with oligonucleotides H1 and H2, respectively.For Library YS-A, diversity was introduced into CDR-H3 with an equimolarmixture of oligonucleotides. For library YS-B, diversity was introducedinto CDR-H3 with an equimolar mixture of oligonucleotides. For libraryYS-B, diversity was introduced into CDR-L3 with an equimolar mixture ofoligonucleotides. The mutagenic oligonucleotides for all CDRs to berandomized were incorporated simultaneously in a single mutagenesisreaction, so that simultaneous incorporation of all the mutagenicoligonucleotides resulted in the introduction of the designed diversityat each position and simultaneously repaired all the TAA stop codons,thus generating an open reading frame that encoded a Fab library memberfused to a homodimerizing leucine zipper and P3C.

The mutagenesis reactions were electroporated into E. coli SS320 (Sidhuet al., supra), and the transformed cells were grown overnight in thepresence of M13-KO7 helper phage (New England Biolabs, Beverly, Mass.)to produce phage particles that encapsulated the phagemid DNA anddisplayed Fab fragments on their surfaces. Each library containedgreater than 5×10⁹ unique members.

(b) Selection of Specific Antibodies from the Naïve Libraries YS-A andYS-B

Phage from library YS-A or YS-B were cycled through rounds of bindingselection to enrich for clones binding to targets of interest. Targetproteins, human VEGF₈₋₁₀₉ and murine VEGF were analyzed separately witheach library. The binding selections were conducted using previouslydescribed methods (Sidhu et al., supra).

NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. withcapture target (5 μg/mL) and blocked for 2 h with Superblock TBS(tris-buffered saline) (Pierce). After overnight growth at 37° C., phagewere concentrated by precipitation with PEG/NaCl and resuspended inSuperblock TBS, 0.05% Tween 20 (Sigma), as described previously (Sidhuet al., supra). Phage solutions (˜10¹² phage/mL) were added to thecoated immunoplates. Following a 2 h incubation to allow for phagebinding, the plates were washed 10 times with PBS, 0.05% Tween 20. Boundphage were eluted with 0.1 M HCl for 10 min and the eluant wasneutralized with 1.0 M Tris base. Eluted phage were amplified in E. coliXL1-blue and used for further rounds of selection.

The libraries were subjected to 5 rounds of selection against eachtarget protein, and at each round, titers were obtained for phagebinding to either the target protein or blank wells coated withSuperblock TBS. The titer of phage bound to target-coated wells dividedby the titer of phage bound to the blank wells was defined as anenrichment ratio used to quantify specific binding of phage pools to thetarget protein; larger enrichment ratios indicate higher specificbinding. The enrichment ratios were observed after 3, 4, or 5 rounds ofselection.

Individual clones from each round of selection were grown in a 96-wellformat in 500 μL of 2YT broth supplemented with carbenicillin andM13-VCS, and the culture supernatants were used directly in phage ELISAs(Sidhu et al., supra) to detect phage-displayed Fabs that bound toplates coated with target protein but not to plates coated with BSA.Specific binders were defined as those phage clones that exhibited anELISA signal at least 15-fold greater on target-coated plates incomparison with BSA-coated plates. Individual clones were screened after2 rounds of selection for binding to human VEGF or after 5 rounds ofselection for the other target proteins. These data were used tocalculate the percentage of specific binders, and the results for eachlibrary against each target protein. Each library produced bindersagainst each target protein.

Individual clones representing specific binders were subjected to DNAsequence analysis, and the sequences of the randomized CDR positions areshown in FIG. 41. It can be seen that, for each target protein, it waspossible to select specific binders that contained only Tyr or Ser atthe randomized positions (although some non-designed mutations wereobserved, which were likely created during library construction probablydue to impurities in the oligonucleotides). Furthermore, the sequencesof specific binders were unique to the target protein against which theywere selected.

Two of the binders listed in FIG. 41 (hVEGF binder #3 and #18) weretested for their affinity with respect to hVEGF and mVEGF. BIAcore datawas obtained according to Chen et al., J Mol Biol. (1999),293(4):865-881. Briefly, binding affinities of hVEGF binder #3 and #18for hVEGF and mVEGF were calculated from association and dissociationrate constants measured using a BIAcore™-2000 surface plasmon resonancesystem (BIAcore, Inc., Piscataway, N.J.). A biosensor chip was activatedfor covalent coupling of VEGF usingN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's (BIAcore, Inc.,Piscataway, N.J.) instructions. hVEGF or mVEGF was buffer-exchanged into10 mM sodium acetate, pH 4.8 and diluted to approximately 30 mg/ml.Aliquots of VEGF were injected at a flow rate of 2 microL/minute toachieve approximately 200-300 response units (RU) of coupled protein. Asolution of 1 M ethanolamine was injected as a blocking agent. Forkinetics measurements, twofold serial dilutions of Fab were injected inPBS/Tween buffer (0.05% Tween20 in phosphate-buffered saline) at 25° C.at a flow rate of 10 microL/minute. Equilibrium dissociation constants,Kd values from surface plasmon resonance measurements were calculated ask_(off)/k_(on). The BIAcore™ data is summarized below.

hVEGF coated on mVEGF coated the chip on the chip Clone #3 k_(a) (M⁻¹ ·s⁻¹) (on-rate) 1.6 × 10⁸     Not detectable k_(d) (s⁻¹) (off-rate) 7 ×10⁻² Not detectable Kd 46 +/− 17 nM Not detectable (>1 uM) Clone #18k_(a) (M⁻¹ · s⁻¹) (on-rate) 1 × 10⁵   4 × 10⁴ k_(d) (s⁻¹) (off-rate) 8 ×10⁻³ 2 × 10⁻² Kd 64 +/− 7 nM 600 +/− 200 nM

Example 11 Additional Anti-VEGF YS Antibodies

Library Construction and Sorting.

A phagemid designed to display bivalent Fab4D5 on the surface of M13bacteriophage was used to construct libraries, as described above.Oligonucleotide-directed mutagenesis was used to replace CDR positionswith TMT degenerate codons, (M=A/C in equal proportions). The positionschosen for randomization were as follows:

Randomized Positions Library CDRL3 CDRH1 CDRH2 CDRH3 YS-A 28, 30, 31,50, 52, 53, 95-100 replaced 32, 33 54, 56, 58 with 7-20 residues YS-B91-94, 96 28, 30, 31, 50, 52, 53, 95-100a replaced 32, 33 54, 56, 58with 7-15 residues.

In CDR-H3, positions 95 through 100a were replaced with random loops ofall possible lengths ranging from 7 to 20 residues (library A) or 7 to15 residues (library B). Each library contained ˜10¹⁰ unique members,and thus, the actual library diversities were comparable to the maximumnumber of unique sequences encoded by the library designs (4×10⁹).

Phage from the libraries were cycled through rounds of binding selectionwith antigen immobilized on 96-well Maxisorp immunoplates (NUNC) as thecapture target, as described previously (Sidhu, S. S., et al., (2000)Methods Enzymol. 328:333-363). After five rounds of selection, phagewere produced from individual clones grown in a 96-well format and theculture supernatants were used in phage ELISAs to detect specificbinding clones. Specific binding clones were determined to be Fab-phagethat bound to the cognate antigen but did not exhibit detectable bindingto seven other proteins.

Competitive Phage ELISA.

A modified phage ELISA was used to estimate the binding affinities ofFabs (Sidhu, (2000), supra; Deshayes, K., et al., (2002) Chem. Biol.9:495-505). Phage ELISAs were carried out on plates coated with antigen,as described above. Phage displaying antibody fragments were seriallydiluted in PBS, 0.5% (w/v) BSA, 0.1% (v/v) Tween 20, and binding wasmeasured to determine a phage concentration giving ˜50% of the signal atsaturation. A fixed, sub-saturating concentration of phage waspreincubated for 2 hours with serial dilutions of antigen and thentransferred to assay plates coated with antigen. After 15 minutesincubation, the plates were washed with PBS, 0.05% Tween 20 andincubated 30 minutes with horseradish peroxidase/anti-M13 antibodyconjugate (1:5000 dilution) (Pharmacia). The plates were washed,developed with TMB substrate (Kirkegaard and Perry Laboratories),quenched with 1.0 M H₃PO₄, and read spectrophotometrically at 450 nm.The binding affinities of the Fabs were determined as IC₅₀ valuesdefined as the concentration of antigen that blocked 50% of the phagebinding to the immobilized antigen. DNA sequencing of 184 binding clonesrevealed 63 unique sequences shown in FIG. 42. Interestingly, the clonesfrom library B exhibit homology within the selected CDR-L3 and CDR-H3sequences. In contrast, the CDR-H3 sequences of the clones from libraryA exhibit homology amongst themselves but are very different from thesequences from library B. Thus, it appears that the nature of the CDR-L3sequence influenced the selection of CDR-H3 sequences, and as a result,two distinct classes of anti-hVEGF antibodies arose from the twodifferent libraries. The clones were screened by competitive phage ELISAand exhibited IC₅₀ values ranging from approximately 60 nM to greaterthan 5 μM.

Protein Purification and Affinity Analysis.

The three anti-hVEGF clones with the highest estimated affinities (topthree sequences in FIG. 42) were purified as free Fab proteins. Fabproteins were purified from E. coli as described previously (Muler, Y.A., et al., (1998) Structure 6:1153-1167). See FIG. 43 for YS1 Fabsequence. The binding kinetics of the purified Fabs (designated Fab-YS1,Fab-YS2 and Fab-YS3) were studied by surface plasmon resonance. Bindingkinetics were determined by surface plasmon resonance using aBIAcore™-3000 with hVEGF immobilized on CM5 chips at ˜500 responseunits, as described previously (Chen, Y., et al., (1999) J. Mol. Biol.293:865-881). Serial dilutions of Fab proteins were injected, andbinding responses were corrected by subtraction of responses on a blankflow cell. For kinetic analysis, a 1:1 Langmuir model of global fittingsof k_(on) and k_(off) was used. The K_(d) values were determined fromthe ratios of k_(on) and k_(off).

YS1 YS2 YS3 k_(a) (10⁴ · M⁻¹ · s⁻¹) 5 ± 1  6 ± 1 5 ± 1 kd (10⁻³ · s⁻¹)2.8 ± 0.1 11.7 ± 0.4 9.4 ± 0.1 K_(D) (nM) 60 ± 20 220 ± 60 190 ± 40 Fab-YS1 exhibited the highest affinity for hVEGF (K_(d)=60 nM), whilethe other two Fabs bound approximately 5-fold less tightly due to fasteroff rates. The sequences of Fab-YS1 and Fab-YS2 differ in only threepositions, and thus, these three differences account for the improvedaffinity of Fab-YS1 in comparison with Fab-YS2.

Immunohistochemistry.

We next investigated the specificity of Fab-YS1 by using the protein tovisualize VEGF in mammalian cells transfected with a gene encoding forVEGF fused to green fluorescent protein (GFP). Human A673 cellsexpressing murine VEGF-GFP were stained and imaged, as described (Peden,A. A., et al., (2004) J. Cell. Biol. 164:1065-1076). In the plus VEGFpanel, Fab-YS1 was pre-incubated for 5 minutes with a 5-fold excess ofrecombinant VEGF before being incubated with the cells. Theimmunohistochemical staining with Fab-YS1 precisely overlapped with thefluorescence signal from the VEGF-GFP fusion (data not shown).Furthermore, the signal was completely blocked by incubating Fab-YS1with hVEGF prior to the staining.

Immunoprecipitation.

We also conducted immunoprecipitations of endogenous hVEGF and comparedthe performance of Fab-YS1 to that of a highly specific, naturalanti-hVEGF monoclonal antibody (A4.6.1) (Kim, K. J., et al., (1992)Growth Factors 7:53-64). A673 cells were metabolically labelled andimmunoprecipitations were performed from the media, as described (Kim,K. J., et al., supra) using 15 ug of anti-GFP polyclonal antibody(Clontech), Fab-YS1 or monoclonal antibody A4.6.1. The immune complexeswere eluted by boiling and resolved by SDS-PAGE on a 14% acrylamide gelunder reducing conditions. The gel was dried and then exposed to aphosphoimager plate overnight. Both antibodies immunoprecipitated anidentical set of bands that likely represent hVEGF variants generated byalternative mRNA splicing (data not shown). Taken together, theseresults show that Fab-YS1 binds to hVEGF with high affinity andspecificity comparable to that of a natural antibody, even in thecomplex cellular milieu

Example 12 In Vivo Activities of the Anti-VEGF Antibodies

G6-23 Inhibits Neonate Mouse Growth and Survival.

Newborn mice (C57/BL6) were intra-peritoneally (i.p.) injected daily at1 day post-natally with G6-23 IgG (50 mg/kg) or Flt-1 (1-3) Fc (50mg/kg), or appropriate controls, gp120-Fc, PBS or no injection. The bodyweights were measured daily and the survival rate of the mice werecounted. As shown in FIG. 10, G6-23 reduced body weight as potently asmFlt-1 (1-3) Fc, which is a known mVEGF antagonist. Moreover, mousesurvival rates were also equivalent between the two groups.Significantly, the results of G6-23 specifically indicated that mVEGF isrequired for the growth and survival of new born mice, whereas theeffect of Flt-1 (1-3) Fc is less specific since it is known to block notonly mVEGF, but also placental growth factor (P1GF) and VEGF-B.

G6-23 Effectively Inhibits the Growth of Xenograft Tumors in Nude Mice.

KM 12 and SW480, two human colon-rectal cancer cell lines, were grown incell culture first and about 10⁶ cells from each cell line were injectedinto host nude mice. When the tumor reached approximately 100 mm³ insize (1 week after injection), G6-23 or control were injected (10 mg/kg)twice weekly (six nude mice were used for each group). The tumor sizeswere measured until day 13 after antibody injection. As shown in FIG.11, G6-23 was significantly effective in reducing tumor volumes of bothKM12 (left graph) and SW480 (right graph) cell lines.

Gene expressions for both hVEGF and mVEGF were examined in KM12xenograft mice. Samples of tumors and surrounding tissues were extractedand Tagman was used to quantify the gene expression levels. In thesexenograft models, hVEGF came from the implanted human KM12 tumor cells,whereas mVEGF came from surrounding host stromal cells. As shown in FIG.12, samples from mice treated with G6-23 on day 3 and day 13 had highergene expression levels for both hVEGF and mVEGF compared to the controlgroups. The results indicate that while mice treated with G6-23 hadreduced tumor growth and much decreased vascularity, expressions of bothmVEGF and hVEGF were up-regulated in response to the reduction ofangiogenesis. It also indicates that at the tumor site, there issignificant infiltration of mouse stromal cells, which is a major sourceof VEGF for the tumor angiogenesis. Therefore, in a preclinical animalmodel such as the xenograft model described herein, an antibody capableof cross-reacting and blocking both hVEGF and mVEGF is necessary forstudying its efficacy.

Mouse (Lewis) lung carcinoma (LL2) cells were also used in a nude micemodel to test the inhibitory effect of G6-23. About 10⁶ LL2 cells in amatrigel formulation were administered subcutaneously in the flank of5-week old beige nude mice. One group of six mice were then treated withG6-23 at 10 mg/kg, injected via i.p. twice weekly for a span of 19 days.Other control agents (i.e., mFlt(1-3)-IgG, rag-10) were also used totreat groups of six mice. As shown in FIG. 13, G6-23 significantlyreduced the rate of tumor growth with a pharmacological effectcomparable to that of mFlt(1-3)-IgG, which is known to be multi-potentin blocking not only mVEGF, but also other angiogenic factors mPlGF andmVEGF-B. Serum levels of bioactive G6-23 was also measured. The resultindicates that its levels (62-121 ug/ml) are well within the expectedrange for a therapeutic neutralizing anti-VEGF antibody.

HM-7 cells (American Type Culture Collection) were also used to studythe tumor growth inhibition in nude mice. G6 IgG antibody, G6-31 IgGantibody, the Avastin™ antibody, and the Y0317 IgG antibody used in thisstudy were expressed in and purified from CHO cells. HM-7 cells weremaintained in culture with F12:DMEM medium, supplemented with 10% FBSand 1% penicillin-streptomycin and 1% Glutamine. Cells were grown at 37°C. in 5% CO2 until confluence, harvested, counted, washed andresuspended in sterile Metrigel at a concentration of 25×10⁶ cells perml. Xenografts were established in 4- to 6-week-old female Beige NudeXID mice by injecting 5×10⁶ of HM-7 cultured cells into the dorsal flankof the mice and allowed to grow. After 48 hours, the tumors werepalpable in all mice, and cohorts were randomly selected (n=10) toprovide day-0 controls. The remaining mice were divided into 23 groupsand injected twice weekly with different anti-VEGF antibodies. Thetreatments for the study groups are as follows: Group A (n=10×1): micetreated with control antibody MAB (an anti-ragweed antibody) in 0.1 mlby interperitoneal injection twice/week with a high dose (5 mg/kg).Group B (n=10×5): mice treated with G6 IgG antibody in 0.1 ml byinterperitoneal injection twice/week with the same dose (0.1, 0.25, 0.5,2 or 5 mg/kg). Group C (n=10×5): mice treated with Y0317 IgG antibody in0.1 ml by interperitoneal injection twice/week with the same dose (0.1,0.25, 0.5, 2 or 5 mg/kg). Group D (n=10×5): mice treated with theAvastin™ antibody in 0.1 ml by interperitoneal injection twice/week withthe same dose (0.1, 0.25, 0.5, 2 or 5 mg/kg). Group E (n=10×5): micetreated with G6-31 IgG antibody in 0.1 ml by interperitoneal injectiontwice/week with the same dose (0.1, 0.25, 0.5, 2 or 5 mg/kg). The mice(n=10 control and treated animals) were killed at days 4, 7, 11, 14, 17and 21 after initiation of injections, and the tumors were excised andweighed.

The results show that there was a significant suppression of tumorgrowth when the G6, G6-31, Y0317 and the Avastin™ antibodies wereadministered (p<0.5) (FIGS. 33A-E). The excised tissues from theanti-VEGF antibody treated mice were smaller in size and lessvascularized as compared to the tumors excised from the control mice. Asdiscussed above, the G6 and the G6-23 antibody unlike the Avastin™antibody and the Y0317 antibody can bind to both human VEGF and mouseVEGF, including mouse stromal VEGF which can be upregulated uponimplantation of human colorectal tumors in mouse models. Directcomparison of the activity of the G6 and G6-31 antibodies, whichantibodies bind similar epitopes, indicates that at most datapoints theantibody with the higher affinity for VEGF-A, the G6-31 antibody, hadincreased tumor growth inhibiting properties.

What is claimed is:
 1. A method of inhibiting vascular permeability in ahuman or a mouse, said method comprising the step of administering tothe human or mouse an isolated monoclonal antibody, or Fab fragmentthereof, wherein the antibody or Fab fragment thereof, is capable ofbinding to human and mouse VEGF and inhibiting the binding of VEGF to aVEGF receptor, and wherein the antibody binds to an epitope comprisingresidues F17, Y21, and Y25 or an epitope comprising residues F17, I83,and Q89 of human VEGF.
 2. The method of claim 1, wherein the antibodybinds to an epitope comprising residues F17, Y21, and Y25 of human VEGFand wherein the epitope further comprises one or more additionalresidues selected from the group consisting of residues M18, Q22, K48,D63, L66, M81, I83, H86, Q89, I91, K101, E103, C104, and P106 of humanVEGF.
 3. The method of claim 1, wherein the antibody binds to an epitopecomprising residues F17, I83, and Q89 of human VEGF and wherein theepitope further comprises one or more additional residues selected fromthe group consisting of residues Y21, Q22, Y25, and D63 of human VEGF.4. A method of inhibiting vascular permeability in a human or a mouse,said method comprising the step of administering to the human or mousean isolated monoclonal antibody, or Fab fragment thereof, wherein theantibody, or Fab fragment thereof, comprises a CDR H1 comprising thecontiguous amino acid sequence DYWIH (SEQ ID NO: 261); a CDR H2comprising the contiguous amino acid sequence GITPAGGYTYYADSVKG (SEQ IDNO: 474), a CDR H3 comprising the contiguous amino acid sequenceFVFFLPYAMDY (SEQ ID NO: 475), a CDR L1 comprising the contiguous aminoacid sequence RASQDVSTAVA (SEQ ID NO: 48), a CDR L2 comprising thecontiguous amino acid sequence SASFLYS (SEQ ID NO: 49), and a CDRL3comprising the contiguous amino acid sequence selected from the groupconsisting of QQSYTTPPT (SEQ ID NO: 363), KQGYANPWT (SEQ IS NO: 143),QQGYGNPFT (SEQ IS NO: 52), and QQGAGSPLT (SEQ ID NO: 68).
 5. The methodof claim 4, wherein the antibody, or Fab fragment thereof, comprises thefollowing amino acid sequence as its heavy chain variable domain (VH):(SEQ ID NO: 31) EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH.


6. The method of claim 4 or 5, wherein the antibody or Fab fragmentthereof, comprises the following amino acid sequence as its light chainvariable domain (VL): (SEQ ID NO. 28)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC.


7. The method of claim 4 or 5, wherein the antibody or Fab fragmentthereof, comprises the following amino acid sequence as its light chainvariable domain (VL): (SEQ ID NO. 30)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQGYANPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC.


8. The method of claim 4 or 5, wherein the antibody or Fab fragmentthereof, comprises the following amino acid sequence as its light chainvariable domain (VL): (SEQ ID NO: 32)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYGNPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC.


9. The method of claim 4 or 5, wherein the antibody or Fab fragmentthereof, comprises the following amino acid sequence as its light chainvariable domain (VL): (SEQ ID NO: 33)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGAGSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC.


10. The method of any one of claims 1, 4, or 5, wherein the human ormouse is suffering from cancer or a disease caused by ocularneovascularization.
 11. The method of claim 10, wherein the cancer isselected from the group consisting of breast cancer, colorectal cancer,non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cancer,prostate cancer, liver cancer, head and neck cancer, melanoma, ovariancancer, mesothelioma, glioblastoma, and multiple myeloma.
 12. The methodof claim 10, wherein the disease caused by ocular neovascularizationcomprises diabetic blindness, retinopathy, primary diabetic retinopathy,age-induced macular degeneration, or rubeosis.
 13. The method of claim1, 4, or 5, wherein the method further comprises the step ofadministering a second therapeutic agent simultaneously or sequentiallywith the isolated monoclonal antibody.
 14. The method of claim 13,wherein the second therapeutic agent is an agent selected from the groupconsisting of an anti-angiogenic agent, an anti-neoplastic composition,a chemotherapeutic agent and a cytotoxic agent.
 15. The method of claim14, wherein the anti-angiogenic agent is an anti-hVEGF antibody capableof binding to the same VEGF epitope as the antibody A4.6.1.